# 4199 inhibition of nfκb-inducing kinase (nik ... · matthias versele1, lut janssen1, tamara...

Matthias Versele1 Lut Janssen1 Tamara Geerts1 Wim Floren1 Boud Janssen1 Hillary Millar1 Edgar Jacoby1 Gerhard Gross1 Yannick Ligny2 Yvan Simonnet2 Nathalie Amblard2 Olivier Querolle2 Imre Csoka2 Virginie Poncelet2 Virginie Tronel2 Sophie Nocquet-Thibault2 Lieven Meerpoel1 James E Edwards4 Mark Salvati3 Tine Casneuf1 Sriram Balasubramanian3 Laurie Lenox3 Charles Theuer5 Ricardo Attar3 and Ian Stansfield2

Janssen Research and Development Beerse Belgium1 Val-de-Reuil France2Spring House PA3 San Diego California4 TRACON Pharmaceuticals Inc San Diego California5

Enhanced NFκB signaling is a hallmark of aggressive lymphoidmalignancies including multiple myeloma (MM) Non-canonical NFκBsignaling involves NIK-dependent activation of IKKα which triggersnuclear accumulation of p52RelB heterodimers NIK is a highlyunstable protein and degradation is mediated by a ubiquitin ligasecomplex consisting of TRAF2 TRAF3 and c-IAP12 (encoded byBIRC23) In a subset of MM NIK is stabilized by mutations in NIKTRAF23 or BIRC23 Here we report on the first potent orallybioavailable NIK kinase inhibitor TRC694 potently inhibits phospho-IKKα prevents nuclear accumulation of p52RelB (but not canonicalNFκB) and represses the associated NFκB gene program selectively inMM cell lines with genetic activation of the non-canonical NFκBpathway Proliferation of NIK translocated TRAF3 or BIRC3 mutantMM cell lines is preferentially inhibited by TRC694 over MM cell lineswhich lack genetic activation of non-canonical NFκB Consistentlyelevated expression of a previously described 11-gene NFκB signatureis predictive of sensitivity to TRC694 in MM A single oral dose ofTRC694 to mice bearing a NIK-translocated MM tumor inhibitsphospho-IKKα and represses p52-mediated transcription of NFκBregulated genes Daily oral dosing of TRC694 completely inhibitsexpansion of NIK or TRAF3 mutant multiple myeloma tumors with nosigns of toxicities in these mouse models In conclusion TRC694provides the first opportunity to test the clinical relevance of non-canonical NFκB inhibition in aggressive lymphoid malignancies

INHIBITION OF NFκB-INDUCING KINASE (NIK) SELECTIVELY ABROGATES NIK AND TRAF3 MUTANT MULTIPLE MYELOMA TUMOR GROWTH

ABSTRACT RESULTS

CONCLUSIONS

bull Genetic alterations (NIK TRAF23 BIRC23) leading to NIK protein stabilization and activation of non-canonical NFκB are found in aggressive B-cell malignancies

bull TRC694 is a potent and selective first-in-class orally bioavailable small molecule NIK inhibitor that inhibits the growth of B-cell malignancies with activation of the non-canonical NFκB pathway in vitro and in vivo

NIK is Critical for Non-Canonical NFκB Activation

BACKGROUND

4199

bull NFkB Inducing Kinase (NIK)ndash essential for non-canonical

NFkB (p52RelB) activation

ndash critical for B-cell maturation

bull NIK-deficient mice ndash viable but reduced B-cell

numbersndash disrupted 2ndary lymphoid

organ architecturendash reduced Ig production

bull NIK activityndash controlled through protein

stability (TRAF23-cIAP12 E3 ubiquitin ligase)

Krappmann amp Vincendeau 2016

NON-CANONICAL NFκB SIGNALINGCANONICAL NFκB SIGNALING

NIK is Activated in Aggressive B-cell Malignancies

Annunziata et alKeats et al Canc Cell 2007 Rahal et al Nat Med 2014 Bea et al PNAS 2013 Zhang et al Cell Rep 2015 Bushell et al Blood 2015 Pham et al Blood 2011 Rossi et al Blood 2012 Ranuncolo et al Blood 2012

Target Profile TRC694

NIK kinase inhibition (IC50) lt10 nM lt 5 nM

NIK affinity and residencetime (Proteros)

lt10 nM

Extended residence time (min)

lt 5 nM

gt 100 nM

Kinase selectivity (DiscoverX) lt10 kinome at 100x NIK Kd

Kd (Nik) lt 1 nM

32409 kinases with 100x Kd

Cellular inhibition of pIKKα or pIkBα

lt100nM pIKKα in L363

lt100nM pIkBα in L363

lt 5 nM

lt 10 nMNIKTRAF3 mutant MMMCL 7day cell proliferation(median IC50)

lt100nM (JJN3 MAVER1) lt 10 lt 10 nM

Selectivity window over WT NIK cell proliferation

gt100-fold (KMS12BM H929 TMD8 OCI-LY3) gt2000 gt 500 gt5000 gt5000 nM

Pharmacology Summary TRC694

TRC694

TRC694 Preferentially Represses NFκB Genes in IGL-NIK

L363 (IGL-NIK MM)

Predefined NFκB 11-gene signature in MM (Annunziata et al 2007)

Gene array 100 nM TRC694 vs DMSO 24h

KMS12-BM (WT-NIK MM)

drawn to same scale (Fold changeSignificance)

Fold change (Log2 scale)

Repression Induction

Potent inhibition of nuclear p52RelB but not canonical NFκB subunit p65 (RelA) in NIK translocated MM

MM cell line Relevant genotype

Median IC50(nM)

RPMI-8226 TRAF3 4

JJN-3 EFTUD2-NIK 9KMS-20 BIRC23 9AMO-1 TRAF3 13MM1R TRAF3 15MM1S TRAF3 20L363 IGL-NIK 23KMS-11 TRAF3 44U266 TRAF3 55MOLP2 BIRC3 59SK-MM-2 WT 79NCI-H929 WT 214KMS-28-PE BIRC23 519LP-1 TRAF3 526JIM-1 WT 891JIM-3 WT 990OPM-2 WT 1579MOLP8 WT 2115ANBL6 TRAF3 4677KMS-12-BM WT 5849KMS-28-BM WT 10000

Expression of NFκB gene signature in MM

TRC694 7-day proliferation assay

Nuclear p52 correlates with TRC694 Sensitivity to the Same Degree as Mutation Status or Activation of a MM

NFκB 11-Gene Signature

mpk

bull Pre-established NIK-translocated MM model (JJN3) Once-daily (qd) oral dosing

bull Efficacy consistent with NFkB gene repression at end of studybull Minimal efficacious dose is 10 mpk qd maximal efficacy seen at gt20

mpk qd (846 TGI with 40 mgkg)

RPMI-8226 (TRAF3) MM1S (TRAF3)

TRC694 is Efficacious in TRAF3 Mutant MM Models

bull Pre-established xenograft model Once-daily oral dosing using PEG400 solution

bull Significant efficacy seen in pre-established TRAF3 mutant xenografts after once daily oral dosing of TRC694 (RPMI-8226 882 TGI MM1S 684 TGI)

bull Efficacy observed (~50 TGI) in TRAF2 mutant IbrR MCL model (Z-138)

Cellular Target Profiling in L363 cell lysates Apparent affinities of TRC694 for various binding partners isolated from L363 cell lysates are displayed

pIKKα inhibition and plasma compound (TRC694) exposure levels after a single oral dose of TRC694 in mice bearing JJN3 (MM) xenografts

NFκB gene repression after a single dose of TRC694 in mice bearing JJN3 xenografts

p52 nuclear exclusion after once daily (qd) or twice daily (bid) dosing of TRC694 for 3 days in mice bearing JJN3 xenografts

Potent inhibition of NIK and repression of p52-mediated NFκB genes upon oral dosing of TRC694 to NIK-translocated-tumor bearing mice

TRC694 is Efficacious in the NIK-translocated JJN-3 Model

B In vivo results

A In vitro selectivity

bull Genetic alterations stabilizing NIK and activating non-canonical NFkB in B-cell malignanciesbull MM ndashNIK BIRC3 (cIAP2) TRAF23 (12-20)

bull MCLndashNIK TRAF23 (17 at diagnosis associated with IbrutinibR) BIRC3 (10)

bull DLBCLndashTRAF3 (9-15)

bull cHL ndashNIK (31)

bull CLL ndashBIRC3 (4 at diagnosis 24 in FludarabineR)

bull Ligandreceptor over-expressionamplification and ligands in bone marrow and lymph node can activate non-canonical NFkBbull MM ndash ~50 of cases show activation of NIK pathway to the same extent as NIK translocated

or TRAF3 mutant cases by NFkB gene signaturebull DLBCL ndash ~30 cases show activation of NIK pathway determined by nuclear p52 IHCbull cHL ndash NIK stabilized in 55 of cases as determined by IHC

bull There are no therapies directly targeting non-canonical NFkB in clinical development

1 0 1 5 2 0 2 50

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

D a y P o s t- im p la n ta t io n

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M) P E G 4 0 0

T R C 6 9 4 _ 1 0 m g k g

T R C 6 9 4 _ 2 0 m g k g

T R C 6 9 4 _ 4 0 m g k g

3 5 4 0 4 5 5 0 5 5 6 00

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0


Me

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T R C 6 9 4 _ 4 0 m g k g

3 0 3 5 3 9 4 3 4 60

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1 5 0 0

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2 5 0 0


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PEG 400

T R C 6 94 _2 0 m g kg

T R C 6 94 _4 0 m g kg

Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
NFkB genes (6h)
100
100
100
100
121156685193
146705024547
75628788642
54477818128
40063617917
77307409851
33510519692
28810385034
49727285997
65906264351
25975593814
32173649083

protocol

BIRC3

ampCLJ150095 raw data RTQ BIRC3

TNFAIP3

ampCLJ150095 results TNFAIP3

NFKB2

ampCLJ150095 NFKB2

RelB

ampCLJ150095 results RelB

GusB

ampCLJ150095 GusB

summary

ampCLJ150095 Summary

summary

vehicle
10 mpk QD
20 mpk QD
40 mpk QD

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
NFkB genes (6h)

Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
100
100
100
100
100
100
100
100
100
100
100
100
1184911292873
42457060582
1043246428844
251503011717
675582299184
337905349034
616378320146
115343368286
47947993562
562826404461
548012911621
581135991695
1037790014802
295939942548
982084932456
156848314092
656644416767
20017142486
883157652966
72380508138
239629499071
240262650446
272994269647
166938074011
574249124853
169989137367
555154778548
65775511458
350689088998
193282389314
763920452803
52211813524
143502566696
158013625919
207094447769
54400087544
96360334555
195937197575
947294167043
210335597586
424648599422
3317708345
827268018455
221344262057
126073015371
23600471615
226127696746
97248127416

protocol

BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev

BIRC3 TNFAIP3 NFKB2 RelB BIRC3 TNFAIP3 NFKB2 RelB

vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data

QTY Mean Ratio control Mean Ratio Vehicle

NFKB2 RelB GusB NFKB2GusB RelBGusB NFKB2 RelB NFKB2 RelB

1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev

NFKB2 RelB NFKB2 RelB

vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data


BIRC-3 TNFAIP3 GusB BIRC3GusB TNFAIP3GusB BIRC3 TNFAIP3 BIRC3 TNFAIP3

1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev

BIRC3 TNFAIP3 BIRC3 TNFAIP3

vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE

Sample Name Detector Name Reporter Task Ct quantity

standaardcurve GusB-St1 FAM GusB FAM Standard 214927 25

GusB-St1 FAM GusB FAM Standard 2177397 25









Slope -33168166 cycleslog decade

Y-Intercept 23322025

R^2 098650163

GusB-NTC FAM GusB FAM Unknown Undetermined


GusB-Sample1 FAM GusB FAM Unknown 2372527


























































































Raw data RelB


standaardcurve RelB-St1 FAM RelB FAM Standard 2698927 25

RelB-St1 FAM RelB FAM Standard 2692717 25











R^2 099032915

RelB-NTC FAM RelB FAM Unknown Undetermined


RelB-Sample1 FAM RelB FAM Unknown 2846331


























































































Raw data NFKB2


standaardcurve NFKB2-St1 FAM NFKB2 FAM Standard 2507928 25

NFKB2-St1 FAM NFKB2 FAM Standard 252904 25












R^2 099819493

NFKB2-NTC FAM NFKB2 FAM Unknown Undetermined


NFKB2-Sample1 FAM NFKB2 FAM Unknown 2816853


























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3


standaardcurve TNFAIP3-St1 FAM TNFAIP3 FAM Standard 2237544 25

TNFAIP3-St1 FAM TNFAIP3 FAM Standard 2227251 25












Y-Intercept 2382267

R^2 0995334

TNFAIP3-NTC FAM TNFAIP3 FAM Unknown Undetermined


TNFAIP3-Sample1 FAM TNFAIP3 FAM Unknown 2352013


























































































Raw data BIRC3


standaardcurve BIRC3-St1 FAM BIRC3 FAM Standard 2133274 25 Automatic Ct Automatic Automatic 1248763

BIRC3-St1 FAM BIRC3 FAM Standard 2125119 25 Automatic Ct Automatic Automatic 1248763












Y-Intercept 227643

R^2 0999058

BIRC3-NTC FAM BIRC3 FAM Unknown Undetermined


BIRC3-Sample1 FAM BIRC3 FAM Unknown 2288865


























































































LJ160012 RTQ PCR JJN-3 PD study

1 Aim

In vivo PD study to test the anti-tumour effect of 64290694-AAA in a subcutaneous JJN-3 xenograft model

Take samples of the mice at 212 and 24 h after treatment

Is there an effect on the NIK-driven genes (BIRC-3 NFKB2 and TNFAIP3)

2 Products

Qiazol Lysis reagent Qiagen 79306

Rneasy mini kit Qiagen 74106

Qiaschreddar Qiagen 79656

QPCR Core kit Eurogentec RT-QP10-05NU

oligodT (12-18) Invitrogen SKU 18418-012 25 microg

superscript II reverse transcriptase invitrogen SKU 18064-071 4x10000 U

10 mM dNTP Mix invitrogen SKU 18427-088 1 ml

DNAse Qiagen QIAG79254

primersetsRTQ PCR

BIRC-3 Applied Biosystems Hs00154109_m1

TNFAIP3 Applied Biosystems Hs00234713_m1

NFKB2 Applied Biosystems Hs00174517_m1

GusB Applied Biosystems Hs00939627_m1

3 Tumorsamples

see protocol OIV154583

4 RTQ PCR

41 Pulverize the tumor samples

httpcovarisinccomcryoprephtm

Pre preparation

- Store the tumor samples at dry ice (plus extra eppendorf 15 ml vials)

- tab off fresh N2

- store the black massive block also in dry ice and let it cool down

- put already in the black massive block the tissueTubes and the glass vials to already cool down

- put the Cryoprep system on

If everything is cold enough then we can start using teh Cryoprep system

Take teh tube with the tumor inside and freeze it in the N2 for several seconds In the mean time with your other hand the glass vials should be screwed onto the tissuetubes

and also frozen in the N2 for several seconds unscrew again the tissuetube from the glass vial Then open the tube and try to get the tumor into the TissueTube

If the tumor is settled in the TissueTube again screw the glass vial unto the TissueTube and put the TissueTube into the liquid N2 for at least 30 s

Then put the whole construction in the machine With the glass vial above and underneath the TissueTube Make sure that the glassvial is not completely tightent to the

TissueTube so that air can escape during the Pulverize

Pulverize at stand 1 for small tumors or stand 2 for bigger tumors (in case of bone kidney or skin tisue use an higher pulverize stand)

Repeat if necessary

Freeze again the TissueTube1 in the liquid N2 and then put everything to on side

Cut at the other side a tip of the TissueTube1 with a pair of scissors and shake everything back into the original sample tube or into a new tube

Freeze the sample back in the liquid N2 and put the samples back into -80degC

42 RNA preparation

- store samples at -80degC

- add 700 microl QIAzol Lysis reagent

- vortex

- add 140 microl chloroform

- vortex

- centrifuge at 6000 g for 15 min at 4degC

after centrifugation the sample seperates into 3 phases upper colorless aqueous phase containing RNA a white interphase and a lower red organic phase

For tissues with an especially high fat content an additional clear phase may be visible below the red organic phase

The volume of the aqueous phase shpuld be approximately 400 microl

- transfer all of the upper aqueous phases to a new eppendorf tube

- add 400 microl 70 EtOH

- mix by pipetting up and down

- put the mix in the Rneasy columns

- spin 15s at max speed at RT

- discard the flow-through

- add 350 microl RW1 buffer to the columns

- spin 15s at high speed

- DNAse treatment

add 10 microl DNAse stock to 70 microl RDD buffer

mix gently by inverting

pipet 80 microl of the DNAse incubation mix directly onto the Rneasy membrane in each well of the plate

seal with Airpore Tape sheet

place at RT for 15 min

remove the Airpore tape

- add 350 microl of buffer RW1 to each column (inhibition of DNAse)

- wait 5 min at RT (to efficiently remove the DNAse digestion)

- centrifuge 15s at max speed at RT


- add 500 microl RPE buffer onto the columns

worksolution add 4 volumes EtOH to the RPE buffer

- spin for 15s at max speed at RT


- add another 500 microl RPE buffer onto the columns



- centrifuge 5 min at max speed to dry the column completely

its important to dry the Rneasy membrane since residual EtOH may interfere with subsequent reactions

- transfer column to a 15 ml collection tube

Do not carry over any flow-trough

- add 30 microl RNAse free water onto the middle of the membrane

- incubate 2 min

- centrifuge for 1 min at max speed to elute the RNA

- repeat the elution with 30 microl RNAse free water to elute the RNA

- measure the concentration of all RNA samples using the Nanodrop8000

if the concentration is not high enough speedvac all the samples during 15 min at 45degC

check the quality of the RNA if 260280 is higher than 2 the quality is OK

- dilute the RNA to 1 microg or somewhere between 400-600 ng

dilute RNA in a volume of 50 microl to equal concentrations

42 cDNA synthesis

- the cDNA synthesis is performed in a PCR machine

- add the following components to a nuclease-free eppendorf tube

- dilute RNA to x ng in DEPC-water

x microg RNA x microl

DEPC-water x microl

11 microl

110 verdunning

ngmicrol 260280 110 800 ng RNAse free water 800 ng add RNAse free water to 11 microl

1 JJN-3 vehicle (PEG400) 2h 2379 2 238 3 8

2 2186 208 219 4 7

3 2741 199 274 3 8

4 12 h 2582 204 258 3 8

5 2450 201 245 3 8

6 1659 209 166 5 6

7 24 h 2005 21 201 4 7

8 1475 215 148 5 6

9 2135 204 214 4 7

10 64290694-AAA 10 mpk QD 2h 2747 199 275 3 8

11 2393 204 239 3 8

12 2211 206 221 4 7

13 12 h 2580 199 258 3 8

14 2985 196 299 3 8

15 2096 207 210 4 7

16 24h 1578 213 158 5 6

17 2483 196 248 3 8

18 2849 198 285 3 8

19 64290694-AAA 20 mpk QD 2h 2074 208 207 4 7

20 2668 203 267 3 8

21 2733 193 273 3 8

22 12 h 2299 205 230 3 8

23 2566 206 257 3 8

24 1237 217 124 6 5

25 24h 2236 202 224 4 7

26 2146 209 215 4 7

27 1489 21 149 5 6

28 64290694-AAA 40 mpk QD 2h 1338 212 134 6 5

29 1504 21 150 5 6

30 3203 188 320 2 9

31 12 h 2374 206 237 3 8

32 2557 204 256 3 8

33 2670 187 267 3 8

34 24h 2598 202 260 3 8

35 2485 205 249 3 8

36 2612 203 261 3 8

37 64290694-AAA 120 mpk QD 2h 1030 106 103 8 3

38 3419 146 342 2 9

39 2379 206 238 3 8

40 12 h 3138 186 314 3 8

41 1834 208 183 4 7

42 1560 211 156 5 6

43 24 h 2881 196 288 3 8

44 2204 208 220 4 7

45 1858 209 186 4 7

- prepare mix A of the following components in a nuclease-free eppendorf tube

per sample

Random Primer (30microgmicrol) 0167 microl

Oligo dT (05 microgml) 1 microl

DEPC-water 233 microl

- add 35 microl from mix A to the diluted RNA (total volume = 145 microl)

- heat to 70degC for 5 min

- quick chill on ice

- collect the contents of the tube by brief centrifugation

- prepare mix B of the following components in a nuclease-free eppendorf tube

per sample

dNTP (20 mM) 05 microl

M-MLV RT 5x buffer 4 microl

M-MLV RT RNAse Point mutant 05 microl


- add 55 microl from mix B to the RNA


- incubate at 20degC for 10 min


- inactivate the reaction by heating at 70degC for 15 min

- store the cDNA at -20degC

43 RTQ PCR using CAS-1200 Corbett Robotics

According SOP Functional Genomics RTQ PCR set-up CAS1200 and Biomek 3000 robot

Functional Genomics RTQ PCR ABI7900HT

per 384-well RTQplate 128 samples can be tested in triplicate (2 times 23 of a 96-well plate)

Mastermix per well

RNAse free water 1815 microl

10x PCR buffer A (Eurogentec) 3 microl

MgCl2 (Eurogentec) 3 microl

dNTP mix 5 mM (Eurogentec) 12 microl

taqman gene expression assays 15 microl

HotGoldStar enzyme 015 microl

27 microl mix + 3 microl cDNA sample

taqman gene expression assays (Applied Biosystems)

BIRC-3 Hs00154109_m1

TNFAIP3 Hs00234713_m1

NFKB2 Hs00174517_m1

housekeeping genes GUS-b Hs99999908_m1

6 Conclusion

There is a nice respression of most of the genes and the respression is the highest 24h after the last dose

6 hvehicle 6 hvehicle 6 hvehicle 6 hvehicle 101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

12 h 12 h 12 h 12 h 25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

24 h 24 h 24 h 24 h 13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

6 h 10 mpk QD 6 h 10 mpk QD 6 h 10 mpk QD 6 h 10 mpk QD 247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

12 h 12 h 12 h 12 h 76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

24 h 24 h 24 h 24 h 78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

6 h20 mpk QD 6 h20 mpk QD 6 h20 mpk QD 6 h20 mpk QD 222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

12 h 12 h 12 h 12 h 66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

24 h 24 h 24 h 24 h 98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529


12 h 12 h 12 h 12 h 18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

24 h 24 h 24 h 24 h 48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991


12 h 12 h 12 h 12 h 137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

24h 24h 24h 24h 12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

226319271451 226319271451 188340381833 188340381833 738673147314 738673147314 1431344305739 1431344305739

32576573983 32576573983 33257839919 33257839919 35441232594 35441232594 66388345401 66388345401

13579208481 13579208481 24462095444 24462095444 12108228094 12108228094 18393683674 18393683674

11988964165 11988964165 12967268992 12967268992 07848466472 07848466472 13722458287 13722458287

Summary

1 Raw data 0345


BIRC-3 TNFAIP3 NFKB2 RelB GusB BIRC3GusB TNFAIP3GusB NFKB2GusB RelBGusB BIRC3 TNFAIP3 NFKB2 RelB BIRC3 TNFAIP3 NFKB2 RelB

1 vehicle (PEG400) 6 h 0097 0159 0345 0813 0114 0849 1395 3028 7138 135 127 237 384 0628 1101 1279 1858

3 2236 4062 4381 4036 3079 0726 1319 1423 1311 116 120 111 71

4 1278 1778 2634 3363 1967 0650 0904 1339 1710 103 82 105 92

5 4893 9615 8983 1197 10143 0482 0948 0886 0118 77 86 69 6

6 3092 6006 3151 3630 5571 0555 1078 0566 0652 88 98 44 35

7 5840 11179 5016 2555 11588 0504 0965 0433 0221 80 88 34 12

11 64290694-AAA 10 mpk QD 0314 0504 0543 1046 3039 0103 0166 0179 0344 16 15 14 19

12 0616 1839 0828 0454 13357 0046 0138 0062 0034 7 13 5 2

15 2320 3581 2163 0319 35091 0066 0102 0062 0009 11 9 5 0

16 0363 0874 0477 0500 4925 0074 0178 0097 0101 12 16 8 5

17 0582 1112 0700 0462 6157 0094 0181 0114 0075 15 16 9 4

18 0541 1533 0504 0323 7449 0073 0206 0068 0043 12 19 5 2

21 64290694-AAA 20 mpk QD 0136 0367 0178 0436 3806 0036 0096 0047 0115 6 9 4 6

22 0084 0309 0194 0136 3349 0025 0092 0058 0041 4 8 5 2

24 0116 0423 0223 0196 4718 0025 0090 0047 0042 4 8 4 2

25 0194 0709 0316 0388 5774 0034 0123 0055 0067 5 11 4 4

26 0127 0446 0237 0288 6879 0018 0065 0034 0042 3 6 3 2

27 0268 0894 0317 0303 19854 0013 0045 0016 0015 2 4 1 1

32 64290694-AAA 40 mpk QD 0173 0344 0238 0489 4757 0036 0072 0050 0103 6 7 4 6

33 0195 0446 0198 0310 4845 0040 0092 0041 0064 6 8 3 3

34 0311 0638 0312 0367 11920 0026 0053 0026 0031 4 5 2 2

35 0107 0363 0137 0315 4570 0023 0079 0030 0069 4 7 2 4

36 0134 0330 0144 0221 5601 0024 0059 0026 0039 4 5 2 2

37 0190 0406 0136 0271 5117 0037 0079 0027 0053 6 7 2 3

2 Mean

control Mean control stdev

BIRC3 TNFAIP3 NFKB2 RelB BIRC3 TNFAIP3 NFKB2 RelB BIRC3 TNFAIP3 NFKB2 RelB

1 vehicle (PEG400) 6 h 135 127 237 384 100 100 100 100 23 19 74 143

3 116 120 111 71

4 103 82 105 92

5 77 86 69 6

6 88 98 44 35

7 80 88 34 12

11 64290694-AAA 10 mpk QD 16 15 14 19 12 15 8 5 3 3 4 7

12 7 13 5 2

15 11 9 5 0

16 12 16 8 5

17 15 16 9 4

18 12 19 5 2

21 64290694-AAA 20 mpk QD 6 9 4 6 4 8 3 3 1 2 1 2

22 4 8 5 2

24 4 8 4 2

25 5 11 4 4

26 3 6 3 2

27 2 4 1 1

32 64290694-AAA 40 mpk QD 6 7 4 6 5 7 3 3 1 1 1 1

33 6 8 3 3

34 4 5 2 2

35 4 7 2 4

36 4 5 2 2

37 6 7 2 3

3 Graph

Mean control stdev

BIRC3 TNFAIP3 NFKB2 RelB BIRC3 TNFAIP3 NFKB2 Relb

vehicle 100 100 100 100 23 19 74 143

10 mpk QD 12 15 8 5 3 3 4 7

20 mpk QD 4 8 3 3 1 2 1 2

40 mpk QD 5 7 3 3 1 1 1 1

Raw data GusB
















R^2 09957464



















































Raw data RelB








RelB-St4 FAM RelB FAM Standard Undetermined 0039063



Y-Intercept 3140816

R^2 09669834



















































Raw data NFKB2















Y-Intercept 2611261

R^2 09981058



















































Raw data TNFAIP3
















R^2 0997717



















































Raw data BIRC3


standaardcurve 1 BIRC3-St1 FAM BIRC3 FAM Standard 2110693 25 Automatic Ct Automatic Automatic 1248763

2 BIRC3-St1 FAM BIRC3 FAM Standard 2120984 25 Automatic Ct Automatic Automatic 1248763









Automatic Ct Automatic Automatic 1248763



Y-Intercept 2266448

R^2 09992404



















































LJ150095 RTQ PCR JJN-3 efficacy study

1 Aim

In vivo efficacy study to test the anti-tumour effect of 64290694-AAA in a subcutaneous JJN-3 xenograft model

Take samples of the mice at 6h after treatment


2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 6 h 4987 203 499 2 9

3 2189 217 219 4 7

4 1796 217 180 4 7

5 4665 206 466 2 9

6 2180 216 218 4 7

7 3386 215 339 2 9

11 64290694-AAA 10 mpk QD 3183 215 318 3 8

12 2569 217 257 3 8

15 3985 213 399 2 9

16 676 217 68 12 -1

17 4704 206 470 2 9

18 2644 217 264 3 8

21 64290694-AAA 20 mpk QD 1455 218 145 5 6

22 3482 215 348 2 9

24 902 217 90 9 2

25 3255 216 326 2 9

26 2316 217 232 3 8

27 3424 213 342 2 9

32 64290694-AAA 40 mpk QD 690 219 69 12 -1

33 1922 218 192 4 7

34 2207 216 221 4 7

35 2185 217 218 4 7

36 155 214 15 5 6

37 67 203 7 12 -1


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion

JNJ-64290694-AAA (PEG400 solution) has a significant anti-tumor efficacy in the JJN-3 xenograft model

The effect on the genes is complet in line with what we expected

The efficacy and level of PD shutdown seems to be better with PEG400 dosing compared to

suspension dosing

vehicle vehicle vehicle vehicle 226319271451 226319271451 188340381833 188340381833 738673147314 738673147314 1431344305739 1431344305739

10 mpk QD 10 mpk QD 10 mpk QD 10 mpk QD 32576573983 32576573983 33257839919 33257839919 35441232594 35441232594 66388345401 66388345401



Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
NFkB genes (6h)
100
100
100
100
121156685193
146705024547
75628788642
54477818128
40063617917
77307409851
33510519692
28810385034
49727285997
65906264351
25975593814
32173649083

protocol

BIRC3


TNFAIP3


NFKB2

ampCLJ150095 NFKB2

RelB


GusB

ampCLJ150095 GusB

summary


summary

vehicle
10 mpk QD
20 mpk QD
40 mpk QD

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
NFkB genes (6h)

Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
100
100
100
100
100
100
100
100
100
100
100
100
1184911292873
42457060582
1043246428844
251503011717
675582299184
337905349034
616378320146
115343368286
47947993562
562826404461
548012911621
581135991695
1037790014802
295939942548
982084932456
156848314092
656644416767
20017142486
883157652966
72380508138
239629499071
240262650446
272994269647
166938074011
574249124853
169989137367
555154778548
65775511458
350689088998
193282389314
763920452803
52211813524
143502566696
158013625919
207094447769
54400087544
96360334555
195937197575
947294167043
210335597586
424648599422
3317708345
827268018455
221344262057
126073015371
23600471615
226127696746
97248127416

protocol

BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































Raw data BIRC3















Y-Intercept 227643

R^2 0999058






























































































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 2h 2379 2 238 3 8

2 2186 208 219 4 7

3 2741 199 274 3 8

4 12 h 2582 204 258 3 8

5 2450 201 245 3 8

6 1659 209 166 5 6

7 24 h 2005 21 201 4 7

8 1475 215 148 5 6

9 2135 204 214 4 7

10 64290694-AAA 10 mpk QD 2h 2747 199 275 3 8

11 2393 204 239 3 8

12 2211 206 221 4 7

13 12 h 2580 199 258 3 8

14 2985 196 299 3 8

15 2096 207 210 4 7

16 24h 1578 213 158 5 6

17 2483 196 248 3 8

18 2849 198 285 3 8

19 64290694-AAA 20 mpk QD 2h 2074 208 207 4 7

20 2668 203 267 3 8

21 2733 193 273 3 8

22 12 h 2299 205 230 3 8

23 2566 206 257 3 8

24 1237 217 124 6 5

25 24h 2236 202 224 4 7

26 2146 209 215 4 7

27 1489 21 149 5 6

28 64290694-AAA 40 mpk QD 2h 1338 212 134 6 5

29 1504 21 150 5 6

30 3203 188 320 2 9

31 12 h 2374 206 237 3 8

32 2557 204 256 3 8

33 2670 187 267 3 8

34 24h 2598 202 260 3 8

35 2485 205 249 3 8

36 2612 203 261 3 8

37 64290694-AAA 120 mpk QD 2h 1030 106 103 8 3

38 3419 146 342 2 9

39 2379 206 238 3 8

40 12 h 3138 186 314 3 8

41 1834 208 183 4 7

42 1560 211 156 5 6

43 24 h 2881 196 288 3 8

44 2204 208 220 4 7

45 1858 209 186 4 7


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion



12 h 12 h 12 h 12 h 25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

24 h 24 h 24 h 24 h 13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959


12 h 12 h 12 h 12 h 76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

24 h 24 h 24 h 24 h 78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665


12 h 12 h 12 h 12 h 66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

24 h 24 h 24 h 24 h 98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529


12 h 12 h 12 h 12 h 18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

24 h 24 h 24 h 24 h 48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991


12 h 12 h 12 h 12 h 137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

24h 24h 24h 24h 12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

226319271451 226319271451 188340381833 188340381833 738673147314 738673147314 1431344305739 1431344305739

32576573983 32576573983 33257839919 33257839919 35441232594 35441232594 66388345401 66388345401

13579208481 13579208481 24462095444 24462095444 12108228094 12108228094 18393683674 18393683674

11988964165 11988964165 12967268992 12967268992 07848466472 07848466472 13722458287 13722458287

Summary

1 Raw data 0345



1 vehicle (PEG400) 6 h 0097 0159 0345 0813 0114 0849 1395 3028 7138 135 127 237 384 0628 1101 1279 1858

3 2236 4062 4381 4036 3079 0726 1319 1423 1311 116 120 111 71

4 1278 1778 2634 3363 1967 0650 0904 1339 1710 103 82 105 92

5 4893 9615 8983 1197 10143 0482 0948 0886 0118 77 86 69 6

6 3092 6006 3151 3630 5571 0555 1078 0566 0652 88 98 44 35

7 5840 11179 5016 2555 11588 0504 0965 0433 0221 80 88 34 12

11 64290694-AAA 10 mpk QD 0314 0504 0543 1046 3039 0103 0166 0179 0344 16 15 14 19

12 0616 1839 0828 0454 13357 0046 0138 0062 0034 7 13 5 2

15 2320 3581 2163 0319 35091 0066 0102 0062 0009 11 9 5 0

16 0363 0874 0477 0500 4925 0074 0178 0097 0101 12 16 8 5

17 0582 1112 0700 0462 6157 0094 0181 0114 0075 15 16 9 4

18 0541 1533 0504 0323 7449 0073 0206 0068 0043 12 19 5 2

21 64290694-AAA 20 mpk QD 0136 0367 0178 0436 3806 0036 0096 0047 0115 6 9 4 6

22 0084 0309 0194 0136 3349 0025 0092 0058 0041 4 8 5 2

24 0116 0423 0223 0196 4718 0025 0090 0047 0042 4 8 4 2

25 0194 0709 0316 0388 5774 0034 0123 0055 0067 5 11 4 4

26 0127 0446 0237 0288 6879 0018 0065 0034 0042 3 6 3 2

27 0268 0894 0317 0303 19854 0013 0045 0016 0015 2 4 1 1

32 64290694-AAA 40 mpk QD 0173 0344 0238 0489 4757 0036 0072 0050 0103 6 7 4 6

33 0195 0446 0198 0310 4845 0040 0092 0041 0064 6 8 3 3

34 0311 0638 0312 0367 11920 0026 0053 0026 0031 4 5 2 2

35 0107 0363 0137 0315 4570 0023 0079 0030 0069 4 7 2 4

36 0134 0330 0144 0221 5601 0024 0059 0026 0039 4 5 2 2

37 0190 0406 0136 0271 5117 0037 0079 0027 0053 6 7 2 3

2 Mean



1 vehicle (PEG400) 6 h 135 127 237 384 100 100 100 100 23 19 74 143

3 116 120 111 71

4 103 82 105 92

5 77 86 69 6

6 88 98 44 35

7 80 88 34 12

11 64290694-AAA 10 mpk QD 16 15 14 19 12 15 8 5 3 3 4 7

12 7 13 5 2

15 11 9 5 0

16 12 16 8 5

17 15 16 9 4

18 12 19 5 2

21 64290694-AAA 20 mpk QD 6 9 4 6 4 8 3 3 1 2 1 2

22 4 8 5 2

24 4 8 4 2

25 5 11 4 4

26 3 6 3 2

27 2 4 1 1

32 64290694-AAA 40 mpk QD 6 7 4 6 5 7 3 3 1 1 1 1

33 6 8 3 3

34 4 5 2 2

35 4 7 2 4

36 4 5 2 2

37 6 7 2 3

3 Graph

Mean control stdev


vehicle 100 100 100 100 23 19 74 143

10 mpk QD 12 15 8 5 3 3 4 7

20 mpk QD 4 8 3 3 1 2 1 2

40 mpk QD 5 7 3 3 1 1 1 1

Raw data GusB
















R^2 09957464



















































Raw data RelB











Y-Intercept 3140816

R^2 09669834



















































Raw data NFKB2















Y-Intercept 2611261

R^2 09981058



















































Raw data TNFAIP3
















R^2 0997717



















































Raw data BIRC3















Y-Intercept 2266448

R^2 09992404




















































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 6 h 4987 203 499 2 9

3 2189 217 219 4 7

4 1796 217 180 4 7

5 4665 206 466 2 9

6 2180 216 218 4 7

7 3386 215 339 2 9

11 64290694-AAA 10 mpk QD 3183 215 318 3 8

12 2569 217 257 3 8

15 3985 213 399 2 9

16 676 217 68 12 -1

17 4704 206 470 2 9

18 2644 217 264 3 8

21 64290694-AAA 20 mpk QD 1455 218 145 5 6

22 3482 215 348 2 9

24 902 217 90 9 2

25 3255 216 326 2 9

26 2316 217 232 3 8

27 3424 213 342 2 9

32 64290694-AAA 40 mpk QD 690 219 69 12 -1

33 1922 218 192 4 7

34 2207 216 221 4 7

35 2185 217 218 4 7

36 155 214 15 5 6

37 67 203 7 12 -1


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion




suspension dosing

vehicle vehicle vehicle vehicle 226319271451 226319271451 188340381833 188340381833 738673147314 738673147314 1431344305739 1431344305739




protocol

BIRC3


TNFAIP3


NFKB2

ampCLJ150095 NFKB2

RelB


GusB

ampCLJ150095 GusB

summary


summary

vehicle
10 mpk QD
20 mpk QD
40 mpk QD

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
NFkB genes (6h)

Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
100
100
100
100
100
100
100
100
100
100
100
100
1184911292873
42457060582
1043246428844
251503011717
675582299184
337905349034
616378320146
115343368286
47947993562
562826404461
548012911621
581135991695
1037790014802
295939942548
982084932456
156848314092
656644416767
20017142486
883157652966
72380508138
239629499071
240262650446
272994269647
166938074011
574249124853
169989137367
555154778548
65775511458
350689088998
193282389314
763920452803
52211813524
143502566696
158013625919
207094447769
54400087544
96360334555
195937197575
947294167043
210335597586
424648599422
3317708345
827268018455
221344262057
126073015371
23600471615
226127696746
97248127416

protocol

BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































Raw data BIRC3















Y-Intercept 227643

R^2 0999058






























































































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 2h 2379 2 238 3 8

2 2186 208 219 4 7

3 2741 199 274 3 8

4 12 h 2582 204 258 3 8

5 2450 201 245 3 8

6 1659 209 166 5 6

7 24 h 2005 21 201 4 7

8 1475 215 148 5 6

9 2135 204 214 4 7

10 64290694-AAA 10 mpk QD 2h 2747 199 275 3 8

11 2393 204 239 3 8

12 2211 206 221 4 7

13 12 h 2580 199 258 3 8

14 2985 196 299 3 8

15 2096 207 210 4 7

16 24h 1578 213 158 5 6

17 2483 196 248 3 8

18 2849 198 285 3 8

19 64290694-AAA 20 mpk QD 2h 2074 208 207 4 7

20 2668 203 267 3 8

21 2733 193 273 3 8

22 12 h 2299 205 230 3 8

23 2566 206 257 3 8

24 1237 217 124 6 5

25 24h 2236 202 224 4 7

26 2146 209 215 4 7

27 1489 21 149 5 6

28 64290694-AAA 40 mpk QD 2h 1338 212 134 6 5

29 1504 21 150 5 6

30 3203 188 320 2 9

31 12 h 2374 206 237 3 8

32 2557 204 256 3 8

33 2670 187 267 3 8

34 24h 2598 202 260 3 8

35 2485 205 249 3 8

36 2612 203 261 3 8

37 64290694-AAA 120 mpk QD 2h 1030 106 103 8 3

38 3419 146 342 2 9

39 2379 206 238 3 8

40 12 h 3138 186 314 3 8

41 1834 208 183 4 7

42 1560 211 156 5 6

43 24 h 2881 196 288 3 8

44 2204 208 220 4 7

45 1858 209 186 4 7


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion



12 h 12 h 12 h 12 h 25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

24 h 24 h 24 h 24 h 13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959


12 h 12 h 12 h 12 h 76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

24 h 24 h 24 h 24 h 78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665


12 h 12 h 12 h 12 h 66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

24 h 24 h 24 h 24 h 98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529


12 h 12 h 12 h 12 h 18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

24 h 24 h 24 h 24 h 48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991


12 h 12 h 12 h 12 h 137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

24h 24h 24h 24h 12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

226319271451 226319271451 188340381833 188340381833 738673147314 738673147314 1431344305739 1431344305739

32576573983 32576573983 33257839919 33257839919 35441232594 35441232594 66388345401 66388345401

13579208481 13579208481 24462095444 24462095444 12108228094 12108228094 18393683674 18393683674

11988964165 11988964165 12967268992 12967268992 07848466472 07848466472 13722458287 13722458287

Summary

1 Raw data 0345



1 vehicle (PEG400) 6 h 0097 0159 0345 0813 0114 0849 1395 3028 7138 135 127 237 384 0628 1101 1279 1858

3 2236 4062 4381 4036 3079 0726 1319 1423 1311 116 120 111 71

4 1278 1778 2634 3363 1967 0650 0904 1339 1710 103 82 105 92

5 4893 9615 8983 1197 10143 0482 0948 0886 0118 77 86 69 6

6 3092 6006 3151 3630 5571 0555 1078 0566 0652 88 98 44 35

7 5840 11179 5016 2555 11588 0504 0965 0433 0221 80 88 34 12

11 64290694-AAA 10 mpk QD 0314 0504 0543 1046 3039 0103 0166 0179 0344 16 15 14 19

12 0616 1839 0828 0454 13357 0046 0138 0062 0034 7 13 5 2

15 2320 3581 2163 0319 35091 0066 0102 0062 0009 11 9 5 0

16 0363 0874 0477 0500 4925 0074 0178 0097 0101 12 16 8 5

17 0582 1112 0700 0462 6157 0094 0181 0114 0075 15 16 9 4

18 0541 1533 0504 0323 7449 0073 0206 0068 0043 12 19 5 2

21 64290694-AAA 20 mpk QD 0136 0367 0178 0436 3806 0036 0096 0047 0115 6 9 4 6

22 0084 0309 0194 0136 3349 0025 0092 0058 0041 4 8 5 2

24 0116 0423 0223 0196 4718 0025 0090 0047 0042 4 8 4 2

25 0194 0709 0316 0388 5774 0034 0123 0055 0067 5 11 4 4

26 0127 0446 0237 0288 6879 0018 0065 0034 0042 3 6 3 2

27 0268 0894 0317 0303 19854 0013 0045 0016 0015 2 4 1 1

32 64290694-AAA 40 mpk QD 0173 0344 0238 0489 4757 0036 0072 0050 0103 6 7 4 6

33 0195 0446 0198 0310 4845 0040 0092 0041 0064 6 8 3 3

34 0311 0638 0312 0367 11920 0026 0053 0026 0031 4 5 2 2

35 0107 0363 0137 0315 4570 0023 0079 0030 0069 4 7 2 4

36 0134 0330 0144 0221 5601 0024 0059 0026 0039 4 5 2 2

37 0190 0406 0136 0271 5117 0037 0079 0027 0053 6 7 2 3

2 Mean



1 vehicle (PEG400) 6 h 135 127 237 384 100 100 100 100 23 19 74 143

3 116 120 111 71

4 103 82 105 92

5 77 86 69 6

6 88 98 44 35

7 80 88 34 12

11 64290694-AAA 10 mpk QD 16 15 14 19 12 15 8 5 3 3 4 7

12 7 13 5 2

15 11 9 5 0

16 12 16 8 5

17 15 16 9 4

18 12 19 5 2

21 64290694-AAA 20 mpk QD 6 9 4 6 4 8 3 3 1 2 1 2

22 4 8 5 2

24 4 8 4 2

25 5 11 4 4

26 3 6 3 2

27 2 4 1 1

32 64290694-AAA 40 mpk QD 6 7 4 6 5 7 3 3 1 1 1 1

33 6 8 3 3

34 4 5 2 2

35 4 7 2 4

36 4 5 2 2

37 6 7 2 3

3 Graph

Mean control stdev


vehicle 100 100 100 100 23 19 74 143

10 mpk QD 12 15 8 5 3 3 4 7

20 mpk QD 4 8 3 3 1 2 1 2

40 mpk QD 5 7 3 3 1 1 1 1

Raw data GusB
















R^2 09957464



















































Raw data RelB











Y-Intercept 3140816

R^2 09669834



















































Raw data NFKB2















Y-Intercept 2611261

R^2 09981058



















































Raw data TNFAIP3
















R^2 0997717



















































Raw data BIRC3















Y-Intercept 2266448

R^2 09992404




















































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 6 h 4987 203 499 2 9

3 2189 217 219 4 7

4 1796 217 180 4 7

5 4665 206 466 2 9

6 2180 216 218 4 7

7 3386 215 339 2 9

11 64290694-AAA 10 mpk QD 3183 215 318 3 8

12 2569 217 257 3 8

15 3985 213 399 2 9

16 676 217 68 12 -1

17 4704 206 470 2 9

18 2644 217 264 3 8

21 64290694-AAA 20 mpk QD 1455 218 145 5 6

22 3482 215 348 2 9

24 902 217 90 9 2

25 3255 216 326 2 9

26 2316 217 232 3 8

27 3424 213 342 2 9

32 64290694-AAA 40 mpk QD 690 219 69 12 -1

33 1922 218 192 4 7

34 2207 216 221 4 7

35 2185 217 218 4 7

36 155 214 15 5 6

37 67 203 7 12 -1


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion




suspension dosing

BIRC3


TNFAIP3


NFKB2

ampCLJ150095 NFKB2

RelB


GusB

ampCLJ150095 GusB

summary


summary

vehicle
10 mpk QD
20 mpk QD
40 mpk QD

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
NFkB genes (6h)

Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
100
100
100
100
100
100
100
100
100
100
100
100
1184911292873
42457060582
1043246428844
251503011717
675582299184
337905349034
616378320146
115343368286
47947993562
562826404461
548012911621
581135991695
1037790014802
295939942548
982084932456
156848314092
656644416767
20017142486
883157652966
72380508138
239629499071
240262650446
272994269647
166938074011
574249124853
169989137367
555154778548
65775511458
350689088998
193282389314
763920452803
52211813524
143502566696
158013625919
207094447769
54400087544
96360334555
195937197575
947294167043
210335597586
424648599422
3317708345
827268018455
221344262057
126073015371
23600471615
226127696746
97248127416

protocol

BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































Raw data BIRC3















Y-Intercept 227643

R^2 0999058






























































































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 2h 2379 2 238 3 8

2 2186 208 219 4 7

3 2741 199 274 3 8

4 12 h 2582 204 258 3 8

5 2450 201 245 3 8

6 1659 209 166 5 6

7 24 h 2005 21 201 4 7

8 1475 215 148 5 6

9 2135 204 214 4 7

10 64290694-AAA 10 mpk QD 2h 2747 199 275 3 8

11 2393 204 239 3 8

12 2211 206 221 4 7

13 12 h 2580 199 258 3 8

14 2985 196 299 3 8

15 2096 207 210 4 7

16 24h 1578 213 158 5 6

17 2483 196 248 3 8

18 2849 198 285 3 8

19 64290694-AAA 20 mpk QD 2h 2074 208 207 4 7

20 2668 203 267 3 8

21 2733 193 273 3 8

22 12 h 2299 205 230 3 8

23 2566 206 257 3 8

24 1237 217 124 6 5

25 24h 2236 202 224 4 7

26 2146 209 215 4 7

27 1489 21 149 5 6

28 64290694-AAA 40 mpk QD 2h 1338 212 134 6 5

29 1504 21 150 5 6

30 3203 188 320 2 9

31 12 h 2374 206 237 3 8

32 2557 204 256 3 8

33 2670 187 267 3 8

34 24h 2598 202 260 3 8

35 2485 205 249 3 8

36 2612 203 261 3 8

37 64290694-AAA 120 mpk QD 2h 1030 106 103 8 3

38 3419 146 342 2 9

39 2379 206 238 3 8

40 12 h 3138 186 314 3 8

41 1834 208 183 4 7

42 1560 211 156 5 6

43 24 h 2881 196 288 3 8

44 2204 208 220 4 7

45 1858 209 186 4 7


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion



12 h 12 h 12 h 12 h 25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

24 h 24 h 24 h 24 h 13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959


12 h 12 h 12 h 12 h 76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

24 h 24 h 24 h 24 h 78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665


12 h 12 h 12 h 12 h 66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

24 h 24 h 24 h 24 h 98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529


12 h 12 h 12 h 12 h 18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

24 h 24 h 24 h 24 h 48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991


12 h 12 h 12 h 12 h 137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

24h 24h 24h 24h 12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

226319271451 226319271451 188340381833 188340381833 738673147314 738673147314 1431344305739 1431344305739

32576573983 32576573983 33257839919 33257839919 35441232594 35441232594 66388345401 66388345401

13579208481 13579208481 24462095444 24462095444 12108228094 12108228094 18393683674 18393683674

11988964165 11988964165 12967268992 12967268992 07848466472 07848466472 13722458287 13722458287

Summary

1 Raw data 0345



1 vehicle (PEG400) 6 h 0097 0159 0345 0813 0114 0849 1395 3028 7138 135 127 237 384 0628 1101 1279 1858

3 2236 4062 4381 4036 3079 0726 1319 1423 1311 116 120 111 71

4 1278 1778 2634 3363 1967 0650 0904 1339 1710 103 82 105 92

5 4893 9615 8983 1197 10143 0482 0948 0886 0118 77 86 69 6

6 3092 6006 3151 3630 5571 0555 1078 0566 0652 88 98 44 35

7 5840 11179 5016 2555 11588 0504 0965 0433 0221 80 88 34 12

11 64290694-AAA 10 mpk QD 0314 0504 0543 1046 3039 0103 0166 0179 0344 16 15 14 19

12 0616 1839 0828 0454 13357 0046 0138 0062 0034 7 13 5 2

15 2320 3581 2163 0319 35091 0066 0102 0062 0009 11 9 5 0

16 0363 0874 0477 0500 4925 0074 0178 0097 0101 12 16 8 5

17 0582 1112 0700 0462 6157 0094 0181 0114 0075 15 16 9 4

18 0541 1533 0504 0323 7449 0073 0206 0068 0043 12 19 5 2

21 64290694-AAA 20 mpk QD 0136 0367 0178 0436 3806 0036 0096 0047 0115 6 9 4 6

22 0084 0309 0194 0136 3349 0025 0092 0058 0041 4 8 5 2

24 0116 0423 0223 0196 4718 0025 0090 0047 0042 4 8 4 2

25 0194 0709 0316 0388 5774 0034 0123 0055 0067 5 11 4 4

26 0127 0446 0237 0288 6879 0018 0065 0034 0042 3 6 3 2

27 0268 0894 0317 0303 19854 0013 0045 0016 0015 2 4 1 1

32 64290694-AAA 40 mpk QD 0173 0344 0238 0489 4757 0036 0072 0050 0103 6 7 4 6

33 0195 0446 0198 0310 4845 0040 0092 0041 0064 6 8 3 3

34 0311 0638 0312 0367 11920 0026 0053 0026 0031 4 5 2 2

35 0107 0363 0137 0315 4570 0023 0079 0030 0069 4 7 2 4

36 0134 0330 0144 0221 5601 0024 0059 0026 0039 4 5 2 2

37 0190 0406 0136 0271 5117 0037 0079 0027 0053 6 7 2 3

2 Mean



1 vehicle (PEG400) 6 h 135 127 237 384 100 100 100 100 23 19 74 143

3 116 120 111 71

4 103 82 105 92

5 77 86 69 6

6 88 98 44 35

7 80 88 34 12

11 64290694-AAA 10 mpk QD 16 15 14 19 12 15 8 5 3 3 4 7

12 7 13 5 2

15 11 9 5 0

16 12 16 8 5

17 15 16 9 4

18 12 19 5 2

21 64290694-AAA 20 mpk QD 6 9 4 6 4 8 3 3 1 2 1 2

22 4 8 5 2

24 4 8 4 2

25 5 11 4 4

26 3 6 3 2

27 2 4 1 1

32 64290694-AAA 40 mpk QD 6 7 4 6 5 7 3 3 1 1 1 1

33 6 8 3 3

34 4 5 2 2

35 4 7 2 4

36 4 5 2 2

37 6 7 2 3

3 Graph

Mean control stdev


vehicle 100 100 100 100 23 19 74 143

10 mpk QD 12 15 8 5 3 3 4 7

20 mpk QD 4 8 3 3 1 2 1 2

40 mpk QD 5 7 3 3 1 1 1 1

Raw data GusB
















R^2 09957464



















































Raw data RelB











Y-Intercept 3140816

R^2 09669834



















































Raw data NFKB2















Y-Intercept 2611261

R^2 09981058



















































Raw data TNFAIP3
















R^2 0997717



















































Raw data BIRC3















Y-Intercept 2266448

R^2 09992404



















































TNFAIP3


NFKB2

ampCLJ150095 NFKB2

RelB


GusB

ampCLJ150095 GusB

summary


summary

vehicle
10 mpk QD
20 mpk QD
40 mpk QD

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
NFkB genes (6h)

Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
100
100
100
100
100
100
100
100
100
100
100
100
1184911292873
42457060582
1043246428844
251503011717
675582299184
337905349034
616378320146
115343368286
47947993562
562826404461
548012911621
581135991695
1037790014802
295939942548
982084932456
156848314092
656644416767
20017142486
883157652966
72380508138
239629499071
240262650446
272994269647
166938074011
574249124853
169989137367
555154778548
65775511458
350689088998
193282389314
763920452803
52211813524
143502566696
158013625919
207094447769
54400087544
96360334555
195937197575
947294167043
210335597586
424648599422
3317708345
827268018455
221344262057
126073015371
23600471615
226127696746
97248127416

protocol

BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































Raw data BIRC3















Y-Intercept 227643

R^2 0999058






























































































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 2h 2379 2 238 3 8

2 2186 208 219 4 7

3 2741 199 274 3 8

4 12 h 2582 204 258 3 8

5 2450 201 245 3 8

6 1659 209 166 5 6

7 24 h 2005 21 201 4 7

8 1475 215 148 5 6

9 2135 204 214 4 7

10 64290694-AAA 10 mpk QD 2h 2747 199 275 3 8

11 2393 204 239 3 8

12 2211 206 221 4 7

13 12 h 2580 199 258 3 8

14 2985 196 299 3 8

15 2096 207 210 4 7

16 24h 1578 213 158 5 6

17 2483 196 248 3 8

18 2849 198 285 3 8

19 64290694-AAA 20 mpk QD 2h 2074 208 207 4 7

20 2668 203 267 3 8

21 2733 193 273 3 8

22 12 h 2299 205 230 3 8

23 2566 206 257 3 8

24 1237 217 124 6 5

25 24h 2236 202 224 4 7

26 2146 209 215 4 7

27 1489 21 149 5 6

28 64290694-AAA 40 mpk QD 2h 1338 212 134 6 5

29 1504 21 150 5 6

30 3203 188 320 2 9

31 12 h 2374 206 237 3 8

32 2557 204 256 3 8

33 2670 187 267 3 8

34 24h 2598 202 260 3 8

35 2485 205 249 3 8

36 2612 203 261 3 8

37 64290694-AAA 120 mpk QD 2h 1030 106 103 8 3

38 3419 146 342 2 9

39 2379 206 238 3 8

40 12 h 3138 186 314 3 8

41 1834 208 183 4 7

42 1560 211 156 5 6

43 24 h 2881 196 288 3 8

44 2204 208 220 4 7

45 1858 209 186 4 7


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion



12 h 12 h 12 h 12 h 25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

24 h 24 h 24 h 24 h 13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959


12 h 12 h 12 h 12 h 76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

24 h 24 h 24 h 24 h 78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665


12 h 12 h 12 h 12 h 66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

24 h 24 h 24 h 24 h 98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529


12 h 12 h 12 h 12 h 18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

24 h 24 h 24 h 24 h 48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991


12 h 12 h 12 h 12 h 137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

24h 24h 24h 24h 12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

226319271451 226319271451 188340381833 188340381833 738673147314 738673147314 1431344305739 1431344305739

32576573983 32576573983 33257839919 33257839919 35441232594 35441232594 66388345401 66388345401

13579208481 13579208481 24462095444 24462095444 12108228094 12108228094 18393683674 18393683674

11988964165 11988964165 12967268992 12967268992 07848466472 07848466472 13722458287 13722458287

Summary

1 Raw data 0345



1 vehicle (PEG400) 6 h 0097 0159 0345 0813 0114 0849 1395 3028 7138 135 127 237 384 0628 1101 1279 1858

3 2236 4062 4381 4036 3079 0726 1319 1423 1311 116 120 111 71

4 1278 1778 2634 3363 1967 0650 0904 1339 1710 103 82 105 92

5 4893 9615 8983 1197 10143 0482 0948 0886 0118 77 86 69 6

6 3092 6006 3151 3630 5571 0555 1078 0566 0652 88 98 44 35

7 5840 11179 5016 2555 11588 0504 0965 0433 0221 80 88 34 12

11 64290694-AAA 10 mpk QD 0314 0504 0543 1046 3039 0103 0166 0179 0344 16 15 14 19

12 0616 1839 0828 0454 13357 0046 0138 0062 0034 7 13 5 2

15 2320 3581 2163 0319 35091 0066 0102 0062 0009 11 9 5 0

16 0363 0874 0477 0500 4925 0074 0178 0097 0101 12 16 8 5

17 0582 1112 0700 0462 6157 0094 0181 0114 0075 15 16 9 4

18 0541 1533 0504 0323 7449 0073 0206 0068 0043 12 19 5 2

21 64290694-AAA 20 mpk QD 0136 0367 0178 0436 3806 0036 0096 0047 0115 6 9 4 6

22 0084 0309 0194 0136 3349 0025 0092 0058 0041 4 8 5 2

24 0116 0423 0223 0196 4718 0025 0090 0047 0042 4 8 4 2

25 0194 0709 0316 0388 5774 0034 0123 0055 0067 5 11 4 4

26 0127 0446 0237 0288 6879 0018 0065 0034 0042 3 6 3 2

27 0268 0894 0317 0303 19854 0013 0045 0016 0015 2 4 1 1

32 64290694-AAA 40 mpk QD 0173 0344 0238 0489 4757 0036 0072 0050 0103 6 7 4 6

33 0195 0446 0198 0310 4845 0040 0092 0041 0064 6 8 3 3

34 0311 0638 0312 0367 11920 0026 0053 0026 0031 4 5 2 2

35 0107 0363 0137 0315 4570 0023 0079 0030 0069 4 7 2 4

36 0134 0330 0144 0221 5601 0024 0059 0026 0039 4 5 2 2

37 0190 0406 0136 0271 5117 0037 0079 0027 0053 6 7 2 3

2 Mean



1 vehicle (PEG400) 6 h 135 127 237 384 100 100 100 100 23 19 74 143

3 116 120 111 71

4 103 82 105 92

5 77 86 69 6

6 88 98 44 35

7 80 88 34 12

11 64290694-AAA 10 mpk QD 16 15 14 19 12 15 8 5 3 3 4 7

12 7 13 5 2

15 11 9 5 0

16 12 16 8 5

17 15 16 9 4

18 12 19 5 2

21 64290694-AAA 20 mpk QD 6 9 4 6 4 8 3 3 1 2 1 2

22 4 8 5 2

24 4 8 4 2

25 5 11 4 4

26 3 6 3 2

27 2 4 1 1

32 64290694-AAA 40 mpk QD 6 7 4 6 5 7 3 3 1 1 1 1

33 6 8 3 3

34 4 5 2 2

35 4 7 2 4

36 4 5 2 2

37 6 7 2 3

3 Graph

Mean control stdev


vehicle 100 100 100 100 23 19 74 143

10 mpk QD 12 15 8 5 3 3 4 7

20 mpk QD 4 8 3 3 1 2 1 2

40 mpk QD 5 7 3 3 1 1 1 1

Raw data GusB
















R^2 09957464



















































Raw data RelB











Y-Intercept 3140816

R^2 09669834



















































Raw data NFKB2















Y-Intercept 2611261

R^2 09981058



















































Raw data TNFAIP3
















R^2 0997717



















































NFKB2

ampCLJ150095 NFKB2

RelB


GusB

ampCLJ150095 GusB

summary


summary

vehicle
10 mpk QD
20 mpk QD
40 mpk QD

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
NFkB genes (6h)

Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
100
100
100
100
100
100
100
100
100
100
100
100
1184911292873
42457060582
1043246428844
251503011717
675582299184
337905349034
616378320146
115343368286
47947993562
562826404461
548012911621
581135991695
1037790014802
295939942548
982084932456
156848314092
656644416767
20017142486
883157652966
72380508138
239629499071
240262650446
272994269647
166938074011
574249124853
169989137367
555154778548
65775511458
350689088998
193282389314
763920452803
52211813524
143502566696
158013625919
207094447769
54400087544
96360334555
195937197575
947294167043
210335597586
424648599422
3317708345
827268018455
221344262057
126073015371
23600471615
226127696746
97248127416

protocol

BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































Raw data BIRC3















Y-Intercept 227643

R^2 0999058






























































































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 2h 2379 2 238 3 8

2 2186 208 219 4 7

3 2741 199 274 3 8

4 12 h 2582 204 258 3 8

5 2450 201 245 3 8

6 1659 209 166 5 6

7 24 h 2005 21 201 4 7

8 1475 215 148 5 6

9 2135 204 214 4 7

10 64290694-AAA 10 mpk QD 2h 2747 199 275 3 8

11 2393 204 239 3 8

12 2211 206 221 4 7

13 12 h 2580 199 258 3 8

14 2985 196 299 3 8

15 2096 207 210 4 7

16 24h 1578 213 158 5 6

17 2483 196 248 3 8

18 2849 198 285 3 8

19 64290694-AAA 20 mpk QD 2h 2074 208 207 4 7

20 2668 203 267 3 8

21 2733 193 273 3 8

22 12 h 2299 205 230 3 8

23 2566 206 257 3 8

24 1237 217 124 6 5

25 24h 2236 202 224 4 7

26 2146 209 215 4 7

27 1489 21 149 5 6

28 64290694-AAA 40 mpk QD 2h 1338 212 134 6 5

29 1504 21 150 5 6

30 3203 188 320 2 9

31 12 h 2374 206 237 3 8

32 2557 204 256 3 8

33 2670 187 267 3 8

34 24h 2598 202 260 3 8

35 2485 205 249 3 8

36 2612 203 261 3 8

37 64290694-AAA 120 mpk QD 2h 1030 106 103 8 3

38 3419 146 342 2 9

39 2379 206 238 3 8

40 12 h 3138 186 314 3 8

41 1834 208 183 4 7

42 1560 211 156 5 6

43 24 h 2881 196 288 3 8

44 2204 208 220 4 7

45 1858 209 186 4 7


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion



12 h 12 h 12 h 12 h 25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

24 h 24 h 24 h 24 h 13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959


12 h 12 h 12 h 12 h 76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

24 h 24 h 24 h 24 h 78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665


12 h 12 h 12 h 12 h 66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

24 h 24 h 24 h 24 h 98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529


12 h 12 h 12 h 12 h 18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

24 h 24 h 24 h 24 h 48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991


12 h 12 h 12 h 12 h 137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

24h 24h 24h 24h 12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

226319271451 226319271451 188340381833 188340381833 738673147314 738673147314 1431344305739 1431344305739

32576573983 32576573983 33257839919 33257839919 35441232594 35441232594 66388345401 66388345401

13579208481 13579208481 24462095444 24462095444 12108228094 12108228094 18393683674 18393683674

11988964165 11988964165 12967268992 12967268992 07848466472 07848466472 13722458287 13722458287

Summary

1 Raw data 0345



1 vehicle (PEG400) 6 h 0097 0159 0345 0813 0114 0849 1395 3028 7138 135 127 237 384 0628 1101 1279 1858

3 2236 4062 4381 4036 3079 0726 1319 1423 1311 116 120 111 71

4 1278 1778 2634 3363 1967 0650 0904 1339 1710 103 82 105 92

5 4893 9615 8983 1197 10143 0482 0948 0886 0118 77 86 69 6

6 3092 6006 3151 3630 5571 0555 1078 0566 0652 88 98 44 35

7 5840 11179 5016 2555 11588 0504 0965 0433 0221 80 88 34 12

11 64290694-AAA 10 mpk QD 0314 0504 0543 1046 3039 0103 0166 0179 0344 16 15 14 19

12 0616 1839 0828 0454 13357 0046 0138 0062 0034 7 13 5 2

15 2320 3581 2163 0319 35091 0066 0102 0062 0009 11 9 5 0

16 0363 0874 0477 0500 4925 0074 0178 0097 0101 12 16 8 5

17 0582 1112 0700 0462 6157 0094 0181 0114 0075 15 16 9 4

18 0541 1533 0504 0323 7449 0073 0206 0068 0043 12 19 5 2

21 64290694-AAA 20 mpk QD 0136 0367 0178 0436 3806 0036 0096 0047 0115 6 9 4 6

22 0084 0309 0194 0136 3349 0025 0092 0058 0041 4 8 5 2

24 0116 0423 0223 0196 4718 0025 0090 0047 0042 4 8 4 2

25 0194 0709 0316 0388 5774 0034 0123 0055 0067 5 11 4 4

26 0127 0446 0237 0288 6879 0018 0065 0034 0042 3 6 3 2

27 0268 0894 0317 0303 19854 0013 0045 0016 0015 2 4 1 1

32 64290694-AAA 40 mpk QD 0173 0344 0238 0489 4757 0036 0072 0050 0103 6 7 4 6

33 0195 0446 0198 0310 4845 0040 0092 0041 0064 6 8 3 3

34 0311 0638 0312 0367 11920 0026 0053 0026 0031 4 5 2 2

35 0107 0363 0137 0315 4570 0023 0079 0030 0069 4 7 2 4

36 0134 0330 0144 0221 5601 0024 0059 0026 0039 4 5 2 2

37 0190 0406 0136 0271 5117 0037 0079 0027 0053 6 7 2 3

2 Mean



1 vehicle (PEG400) 6 h 135 127 237 384 100 100 100 100 23 19 74 143

3 116 120 111 71

4 103 82 105 92

5 77 86 69 6

6 88 98 44 35

7 80 88 34 12

11 64290694-AAA 10 mpk QD 16 15 14 19 12 15 8 5 3 3 4 7

12 7 13 5 2

15 11 9 5 0

16 12 16 8 5

17 15 16 9 4

18 12 19 5 2

21 64290694-AAA 20 mpk QD 6 9 4 6 4 8 3 3 1 2 1 2

22 4 8 5 2

24 4 8 4 2

25 5 11 4 4

26 3 6 3 2

27 2 4 1 1

32 64290694-AAA 40 mpk QD 6 7 4 6 5 7 3 3 1 1 1 1

33 6 8 3 3

34 4 5 2 2

35 4 7 2 4

36 4 5 2 2

37 6 7 2 3

3 Graph

Mean control stdev


vehicle 100 100 100 100 23 19 74 143

10 mpk QD 12 15 8 5 3 3 4 7

20 mpk QD 4 8 3 3 1 2 1 2

40 mpk QD 5 7 3 3 1 1 1 1

Raw data GusB
















R^2 09957464



















































Raw data RelB











Y-Intercept 3140816

R^2 09669834



















































Raw data NFKB2















Y-Intercept 2611261

R^2 09981058



















































RelB


GusB

ampCLJ150095 GusB

summary


summary

vehicle
10 mpk QD
20 mpk QD
40 mpk QD

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
NFkB genes (6h)

Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
100
100
100
100
100
100
100
100
100
100
100
100
1184911292873
42457060582
1043246428844
251503011717
675582299184
337905349034
616378320146
115343368286
47947993562
562826404461
548012911621
581135991695
1037790014802
295939942548
982084932456
156848314092
656644416767
20017142486
883157652966
72380508138
239629499071
240262650446
272994269647
166938074011
574249124853
169989137367
555154778548
65775511458
350689088998
193282389314
763920452803
52211813524
143502566696
158013625919
207094447769
54400087544
96360334555
195937197575
947294167043
210335597586
424648599422
3317708345
827268018455
221344262057
126073015371
23600471615
226127696746
97248127416

protocol

BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































Raw data BIRC3















Y-Intercept 227643

R^2 0999058






























































































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 2h 2379 2 238 3 8

2 2186 208 219 4 7

3 2741 199 274 3 8

4 12 h 2582 204 258 3 8

5 2450 201 245 3 8

6 1659 209 166 5 6

7 24 h 2005 21 201 4 7

8 1475 215 148 5 6

9 2135 204 214 4 7

10 64290694-AAA 10 mpk QD 2h 2747 199 275 3 8

11 2393 204 239 3 8

12 2211 206 221 4 7

13 12 h 2580 199 258 3 8

14 2985 196 299 3 8

15 2096 207 210 4 7

16 24h 1578 213 158 5 6

17 2483 196 248 3 8

18 2849 198 285 3 8

19 64290694-AAA 20 mpk QD 2h 2074 208 207 4 7

20 2668 203 267 3 8

21 2733 193 273 3 8

22 12 h 2299 205 230 3 8

23 2566 206 257 3 8

24 1237 217 124 6 5

25 24h 2236 202 224 4 7

26 2146 209 215 4 7

27 1489 21 149 5 6

28 64290694-AAA 40 mpk QD 2h 1338 212 134 6 5

29 1504 21 150 5 6

30 3203 188 320 2 9

31 12 h 2374 206 237 3 8

32 2557 204 256 3 8

33 2670 187 267 3 8

34 24h 2598 202 260 3 8

35 2485 205 249 3 8

36 2612 203 261 3 8

37 64290694-AAA 120 mpk QD 2h 1030 106 103 8 3

38 3419 146 342 2 9

39 2379 206 238 3 8

40 12 h 3138 186 314 3 8

41 1834 208 183 4 7

42 1560 211 156 5 6

43 24 h 2881 196 288 3 8

44 2204 208 220 4 7

45 1858 209 186 4 7


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion



12 h 12 h 12 h 12 h 25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

24 h 24 h 24 h 24 h 13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959


12 h 12 h 12 h 12 h 76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

24 h 24 h 24 h 24 h 78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665


12 h 12 h 12 h 12 h 66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

24 h 24 h 24 h 24 h 98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529


12 h 12 h 12 h 12 h 18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

24 h 24 h 24 h 24 h 48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991


12 h 12 h 12 h 12 h 137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

24h 24h 24h 24h 12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

226319271451 226319271451 188340381833 188340381833 738673147314 738673147314 1431344305739 1431344305739

32576573983 32576573983 33257839919 33257839919 35441232594 35441232594 66388345401 66388345401

13579208481 13579208481 24462095444 24462095444 12108228094 12108228094 18393683674 18393683674

11988964165 11988964165 12967268992 12967268992 07848466472 07848466472 13722458287 13722458287

Summary

1 Raw data 0345



1 vehicle (PEG400) 6 h 0097 0159 0345 0813 0114 0849 1395 3028 7138 135 127 237 384 0628 1101 1279 1858

3 2236 4062 4381 4036 3079 0726 1319 1423 1311 116 120 111 71

4 1278 1778 2634 3363 1967 0650 0904 1339 1710 103 82 105 92

5 4893 9615 8983 1197 10143 0482 0948 0886 0118 77 86 69 6

6 3092 6006 3151 3630 5571 0555 1078 0566 0652 88 98 44 35

7 5840 11179 5016 2555 11588 0504 0965 0433 0221 80 88 34 12

11 64290694-AAA 10 mpk QD 0314 0504 0543 1046 3039 0103 0166 0179 0344 16 15 14 19

12 0616 1839 0828 0454 13357 0046 0138 0062 0034 7 13 5 2

15 2320 3581 2163 0319 35091 0066 0102 0062 0009 11 9 5 0

16 0363 0874 0477 0500 4925 0074 0178 0097 0101 12 16 8 5

17 0582 1112 0700 0462 6157 0094 0181 0114 0075 15 16 9 4

18 0541 1533 0504 0323 7449 0073 0206 0068 0043 12 19 5 2

21 64290694-AAA 20 mpk QD 0136 0367 0178 0436 3806 0036 0096 0047 0115 6 9 4 6

22 0084 0309 0194 0136 3349 0025 0092 0058 0041 4 8 5 2

24 0116 0423 0223 0196 4718 0025 0090 0047 0042 4 8 4 2

25 0194 0709 0316 0388 5774 0034 0123 0055 0067 5 11 4 4

26 0127 0446 0237 0288 6879 0018 0065 0034 0042 3 6 3 2

27 0268 0894 0317 0303 19854 0013 0045 0016 0015 2 4 1 1

32 64290694-AAA 40 mpk QD 0173 0344 0238 0489 4757 0036 0072 0050 0103 6 7 4 6

33 0195 0446 0198 0310 4845 0040 0092 0041 0064 6 8 3 3

34 0311 0638 0312 0367 11920 0026 0053 0026 0031 4 5 2 2

35 0107 0363 0137 0315 4570 0023 0079 0030 0069 4 7 2 4

36 0134 0330 0144 0221 5601 0024 0059 0026 0039 4 5 2 2

37 0190 0406 0136 0271 5117 0037 0079 0027 0053 6 7 2 3

2 Mean



1 vehicle (PEG400) 6 h 135 127 237 384 100 100 100 100 23 19 74 143

3 116 120 111 71

4 103 82 105 92

5 77 86 69 6

6 88 98 44 35

7 80 88 34 12

11 64290694-AAA 10 mpk QD 16 15 14 19 12 15 8 5 3 3 4 7

12 7 13 5 2

15 11 9 5 0

16 12 16 8 5

17 15 16 9 4

18 12 19 5 2

21 64290694-AAA 20 mpk QD 6 9 4 6 4 8 3 3 1 2 1 2

22 4 8 5 2

24 4 8 4 2

25 5 11 4 4

26 3 6 3 2

27 2 4 1 1

32 64290694-AAA 40 mpk QD 6 7 4 6 5 7 3 3 1 1 1 1

33 6 8 3 3

34 4 5 2 2

35 4 7 2 4

36 4 5 2 2

37 6 7 2 3

3 Graph

Mean control stdev


vehicle 100 100 100 100 23 19 74 143

10 mpk QD 12 15 8 5 3 3 4 7

20 mpk QD 4 8 3 3 1 2 1 2

40 mpk QD 5 7 3 3 1 1 1 1

Raw data GusB
















R^2 09957464



















































Raw data RelB











Y-Intercept 3140816

R^2 09669834



















































GusB

ampCLJ150095 GusB

summary


summary

vehicle
10 mpk QD
20 mpk QD
40 mpk QD

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
NFkB genes (6h)

Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
100
100
100
100
100
100
100
100
100
100
100
100
1184911292873
42457060582
1043246428844
251503011717
675582299184
337905349034
616378320146
115343368286
47947993562
562826404461
548012911621
581135991695
1037790014802
295939942548
982084932456
156848314092
656644416767
20017142486
883157652966
72380508138
239629499071
240262650446
272994269647
166938074011
574249124853
169989137367
555154778548
65775511458
350689088998
193282389314
763920452803
52211813524
143502566696
158013625919
207094447769
54400087544
96360334555
195937197575
947294167043
210335597586
424648599422
3317708345
827268018455
221344262057
126073015371
23600471615
226127696746
97248127416

protocol

BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































Raw data BIRC3















Y-Intercept 227643

R^2 0999058






























































































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 2h 2379 2 238 3 8

2 2186 208 219 4 7

3 2741 199 274 3 8

4 12 h 2582 204 258 3 8

5 2450 201 245 3 8

6 1659 209 166 5 6

7 24 h 2005 21 201 4 7

8 1475 215 148 5 6

9 2135 204 214 4 7

10 64290694-AAA 10 mpk QD 2h 2747 199 275 3 8

11 2393 204 239 3 8

12 2211 206 221 4 7

13 12 h 2580 199 258 3 8

14 2985 196 299 3 8

15 2096 207 210 4 7

16 24h 1578 213 158 5 6

17 2483 196 248 3 8

18 2849 198 285 3 8

19 64290694-AAA 20 mpk QD 2h 2074 208 207 4 7

20 2668 203 267 3 8

21 2733 193 273 3 8

22 12 h 2299 205 230 3 8

23 2566 206 257 3 8

24 1237 217 124 6 5

25 24h 2236 202 224 4 7

26 2146 209 215 4 7

27 1489 21 149 5 6

28 64290694-AAA 40 mpk QD 2h 1338 212 134 6 5

29 1504 21 150 5 6

30 3203 188 320 2 9

31 12 h 2374 206 237 3 8

32 2557 204 256 3 8

33 2670 187 267 3 8

34 24h 2598 202 260 3 8

35 2485 205 249 3 8

36 2612 203 261 3 8

37 64290694-AAA 120 mpk QD 2h 1030 106 103 8 3

38 3419 146 342 2 9

39 2379 206 238 3 8

40 12 h 3138 186 314 3 8

41 1834 208 183 4 7

42 1560 211 156 5 6

43 24 h 2881 196 288 3 8

44 2204 208 220 4 7

45 1858 209 186 4 7


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion



12 h 12 h 12 h 12 h 25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

24 h 24 h 24 h 24 h 13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959


12 h 12 h 12 h 12 h 76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

24 h 24 h 24 h 24 h 78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665


12 h 12 h 12 h 12 h 66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

24 h 24 h 24 h 24 h 98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529


12 h 12 h 12 h 12 h 18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

24 h 24 h 24 h 24 h 48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991


12 h 12 h 12 h 12 h 137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

24h 24h 24h 24h 12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

226319271451 226319271451 188340381833 188340381833 738673147314 738673147314 1431344305739 1431344305739

32576573983 32576573983 33257839919 33257839919 35441232594 35441232594 66388345401 66388345401

13579208481 13579208481 24462095444 24462095444 12108228094 12108228094 18393683674 18393683674

11988964165 11988964165 12967268992 12967268992 07848466472 07848466472 13722458287 13722458287

Summary

1 Raw data 0345



1 vehicle (PEG400) 6 h 0097 0159 0345 0813 0114 0849 1395 3028 7138 135 127 237 384 0628 1101 1279 1858

3 2236 4062 4381 4036 3079 0726 1319 1423 1311 116 120 111 71

4 1278 1778 2634 3363 1967 0650 0904 1339 1710 103 82 105 92

5 4893 9615 8983 1197 10143 0482 0948 0886 0118 77 86 69 6

6 3092 6006 3151 3630 5571 0555 1078 0566 0652 88 98 44 35

7 5840 11179 5016 2555 11588 0504 0965 0433 0221 80 88 34 12

11 64290694-AAA 10 mpk QD 0314 0504 0543 1046 3039 0103 0166 0179 0344 16 15 14 19

12 0616 1839 0828 0454 13357 0046 0138 0062 0034 7 13 5 2

15 2320 3581 2163 0319 35091 0066 0102 0062 0009 11 9 5 0

16 0363 0874 0477 0500 4925 0074 0178 0097 0101 12 16 8 5

17 0582 1112 0700 0462 6157 0094 0181 0114 0075 15 16 9 4

18 0541 1533 0504 0323 7449 0073 0206 0068 0043 12 19 5 2

21 64290694-AAA 20 mpk QD 0136 0367 0178 0436 3806 0036 0096 0047 0115 6 9 4 6

22 0084 0309 0194 0136 3349 0025 0092 0058 0041 4 8 5 2

24 0116 0423 0223 0196 4718 0025 0090 0047 0042 4 8 4 2

25 0194 0709 0316 0388 5774 0034 0123 0055 0067 5 11 4 4

26 0127 0446 0237 0288 6879 0018 0065 0034 0042 3 6 3 2

27 0268 0894 0317 0303 19854 0013 0045 0016 0015 2 4 1 1

32 64290694-AAA 40 mpk QD 0173 0344 0238 0489 4757 0036 0072 0050 0103 6 7 4 6

33 0195 0446 0198 0310 4845 0040 0092 0041 0064 6 8 3 3

34 0311 0638 0312 0367 11920 0026 0053 0026 0031 4 5 2 2

35 0107 0363 0137 0315 4570 0023 0079 0030 0069 4 7 2 4

36 0134 0330 0144 0221 5601 0024 0059 0026 0039 4 5 2 2

37 0190 0406 0136 0271 5117 0037 0079 0027 0053 6 7 2 3

2 Mean



1 vehicle (PEG400) 6 h 135 127 237 384 100 100 100 100 23 19 74 143

3 116 120 111 71

4 103 82 105 92

5 77 86 69 6

6 88 98 44 35

7 80 88 34 12

11 64290694-AAA 10 mpk QD 16 15 14 19 12 15 8 5 3 3 4 7

12 7 13 5 2

15 11 9 5 0

16 12 16 8 5

17 15 16 9 4

18 12 19 5 2

21 64290694-AAA 20 mpk QD 6 9 4 6 4 8 3 3 1 2 1 2

22 4 8 5 2

24 4 8 4 2

25 5 11 4 4

26 3 6 3 2

27 2 4 1 1

32 64290694-AAA 40 mpk QD 6 7 4 6 5 7 3 3 1 1 1 1

33 6 8 3 3

34 4 5 2 2

35 4 7 2 4

36 4 5 2 2

37 6 7 2 3

3 Graph

Mean control stdev


vehicle 100 100 100 100 23 19 74 143

10 mpk QD 12 15 8 5 3 3 4 7

20 mpk QD 4 8 3 3 1 2 1 2

40 mpk QD 5 7 3 3 1 1 1 1

Raw data GusB
















R^2 09957464



















































summary


summary

vehicle
10 mpk QD
20 mpk QD
40 mpk QD

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
NFkB genes (6h)

Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
100
100
100
100
100
100
100
100
100
100
100
100
1184911292873
42457060582
1043246428844
251503011717
675582299184
337905349034
616378320146
115343368286
47947993562
562826404461
548012911621
581135991695
1037790014802
295939942548
982084932456
156848314092
656644416767
20017142486
883157652966
72380508138
239629499071
240262650446
272994269647
166938074011
574249124853
169989137367
555154778548
65775511458
350689088998
193282389314
763920452803
52211813524
143502566696
158013625919
207094447769
54400087544
96360334555
195937197575
947294167043
210335597586
424648599422
3317708345
827268018455
221344262057
126073015371
23600471615
226127696746
97248127416

protocol

BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































Raw data BIRC3















Y-Intercept 227643

R^2 0999058






























































































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 2h 2379 2 238 3 8

2 2186 208 219 4 7

3 2741 199 274 3 8

4 12 h 2582 204 258 3 8

5 2450 201 245 3 8

6 1659 209 166 5 6

7 24 h 2005 21 201 4 7

8 1475 215 148 5 6

9 2135 204 214 4 7

10 64290694-AAA 10 mpk QD 2h 2747 199 275 3 8

11 2393 204 239 3 8

12 2211 206 221 4 7

13 12 h 2580 199 258 3 8

14 2985 196 299 3 8

15 2096 207 210 4 7

16 24h 1578 213 158 5 6

17 2483 196 248 3 8

18 2849 198 285 3 8

19 64290694-AAA 20 mpk QD 2h 2074 208 207 4 7

20 2668 203 267 3 8

21 2733 193 273 3 8

22 12 h 2299 205 230 3 8

23 2566 206 257 3 8

24 1237 217 124 6 5

25 24h 2236 202 224 4 7

26 2146 209 215 4 7

27 1489 21 149 5 6

28 64290694-AAA 40 mpk QD 2h 1338 212 134 6 5

29 1504 21 150 5 6

30 3203 188 320 2 9

31 12 h 2374 206 237 3 8

32 2557 204 256 3 8

33 2670 187 267 3 8

34 24h 2598 202 260 3 8

35 2485 205 249 3 8

36 2612 203 261 3 8

37 64290694-AAA 120 mpk QD 2h 1030 106 103 8 3

38 3419 146 342 2 9

39 2379 206 238 3 8

40 12 h 3138 186 314 3 8

41 1834 208 183 4 7

42 1560 211 156 5 6

43 24 h 2881 196 288 3 8

44 2204 208 220 4 7

45 1858 209 186 4 7


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion



12 h 12 h 12 h 12 h 25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

24 h 24 h 24 h 24 h 13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959


12 h 12 h 12 h 12 h 76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

24 h 24 h 24 h 24 h 78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665


12 h 12 h 12 h 12 h 66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

24 h 24 h 24 h 24 h 98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529


12 h 12 h 12 h 12 h 18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

24 h 24 h 24 h 24 h 48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991


12 h 12 h 12 h 12 h 137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

24h 24h 24h 24h 12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

226319271451 226319271451 188340381833 188340381833 738673147314 738673147314 1431344305739 1431344305739

32576573983 32576573983 33257839919 33257839919 35441232594 35441232594 66388345401 66388345401

13579208481 13579208481 24462095444 24462095444 12108228094 12108228094 18393683674 18393683674

11988964165 11988964165 12967268992 12967268992 07848466472 07848466472 13722458287 13722458287

Summary

1 Raw data 0345



1 vehicle (PEG400) 6 h 0097 0159 0345 0813 0114 0849 1395 3028 7138 135 127 237 384 0628 1101 1279 1858

3 2236 4062 4381 4036 3079 0726 1319 1423 1311 116 120 111 71

4 1278 1778 2634 3363 1967 0650 0904 1339 1710 103 82 105 92

5 4893 9615 8983 1197 10143 0482 0948 0886 0118 77 86 69 6

6 3092 6006 3151 3630 5571 0555 1078 0566 0652 88 98 44 35

7 5840 11179 5016 2555 11588 0504 0965 0433 0221 80 88 34 12

11 64290694-AAA 10 mpk QD 0314 0504 0543 1046 3039 0103 0166 0179 0344 16 15 14 19

12 0616 1839 0828 0454 13357 0046 0138 0062 0034 7 13 5 2

15 2320 3581 2163 0319 35091 0066 0102 0062 0009 11 9 5 0

16 0363 0874 0477 0500 4925 0074 0178 0097 0101 12 16 8 5

17 0582 1112 0700 0462 6157 0094 0181 0114 0075 15 16 9 4

18 0541 1533 0504 0323 7449 0073 0206 0068 0043 12 19 5 2

21 64290694-AAA 20 mpk QD 0136 0367 0178 0436 3806 0036 0096 0047 0115 6 9 4 6

22 0084 0309 0194 0136 3349 0025 0092 0058 0041 4 8 5 2

24 0116 0423 0223 0196 4718 0025 0090 0047 0042 4 8 4 2

25 0194 0709 0316 0388 5774 0034 0123 0055 0067 5 11 4 4

26 0127 0446 0237 0288 6879 0018 0065 0034 0042 3 6 3 2

27 0268 0894 0317 0303 19854 0013 0045 0016 0015 2 4 1 1

32 64290694-AAA 40 mpk QD 0173 0344 0238 0489 4757 0036 0072 0050 0103 6 7 4 6

33 0195 0446 0198 0310 4845 0040 0092 0041 0064 6 8 3 3

34 0311 0638 0312 0367 11920 0026 0053 0026 0031 4 5 2 2

35 0107 0363 0137 0315 4570 0023 0079 0030 0069 4 7 2 4

36 0134 0330 0144 0221 5601 0024 0059 0026 0039 4 5 2 2

37 0190 0406 0136 0271 5117 0037 0079 0027 0053 6 7 2 3

2 Mean



1 vehicle (PEG400) 6 h 135 127 237 384 100 100 100 100 23 19 74 143

3 116 120 111 71

4 103 82 105 92

5 77 86 69 6

6 88 98 44 35

7 80 88 34 12

11 64290694-AAA 10 mpk QD 16 15 14 19 12 15 8 5 3 3 4 7

12 7 13 5 2

15 11 9 5 0

16 12 16 8 5

17 15 16 9 4

18 12 19 5 2

21 64290694-AAA 20 mpk QD 6 9 4 6 4 8 3 3 1 2 1 2

22 4 8 5 2

24 4 8 4 2

25 5 11 4 4

26 3 6 3 2

27 2 4 1 1

32 64290694-AAA 40 mpk QD 6 7 4 6 5 7 3 3 1 1 1 1

33 6 8 3 3

34 4 5 2 2

35 4 7 2 4

36 4 5 2 2

37 6 7 2 3

3 Graph

Mean control stdev


vehicle 100 100 100 100 23 19 74 143

10 mpk QD 12 15 8 5 3 3 4 7

20 mpk QD 4 8 3 3 1 2 1 2

40 mpk QD 5 7 3 3 1 1 1 1

summary

vehicle
10 mpk QD
20 mpk QD
40 mpk QD

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
NFkB genes (6h)

Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
100
100
100
100
100
100
100
100
100
100
100
100
1184911292873
42457060582
1043246428844
251503011717
675582299184
337905349034
616378320146
115343368286
47947993562
562826404461
548012911621
581135991695
1037790014802
295939942548
982084932456
156848314092
656644416767
20017142486
883157652966
72380508138
239629499071
240262650446
272994269647
166938074011
574249124853
169989137367
555154778548
65775511458
350689088998
193282389314
763920452803
52211813524
143502566696
158013625919
207094447769
54400087544
96360334555
195937197575
947294167043
210335597586
424648599422
3317708345
827268018455
221344262057
126073015371
23600471615
226127696746
97248127416

protocol

BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































Raw data BIRC3















Y-Intercept 227643

R^2 0999058






























































































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 2h 2379 2 238 3 8

2 2186 208 219 4 7

3 2741 199 274 3 8

4 12 h 2582 204 258 3 8

5 2450 201 245 3 8

6 1659 209 166 5 6

7 24 h 2005 21 201 4 7

8 1475 215 148 5 6

9 2135 204 214 4 7

10 64290694-AAA 10 mpk QD 2h 2747 199 275 3 8

11 2393 204 239 3 8

12 2211 206 221 4 7

13 12 h 2580 199 258 3 8

14 2985 196 299 3 8

15 2096 207 210 4 7

16 24h 1578 213 158 5 6

17 2483 196 248 3 8

18 2849 198 285 3 8

19 64290694-AAA 20 mpk QD 2h 2074 208 207 4 7

20 2668 203 267 3 8

21 2733 193 273 3 8

22 12 h 2299 205 230 3 8

23 2566 206 257 3 8

24 1237 217 124 6 5

25 24h 2236 202 224 4 7

26 2146 209 215 4 7

27 1489 21 149 5 6

28 64290694-AAA 40 mpk QD 2h 1338 212 134 6 5

29 1504 21 150 5 6

30 3203 188 320 2 9

31 12 h 2374 206 237 3 8

32 2557 204 256 3 8

33 2670 187 267 3 8

34 24h 2598 202 260 3 8

35 2485 205 249 3 8

36 2612 203 261 3 8

37 64290694-AAA 120 mpk QD 2h 1030 106 103 8 3

38 3419 146 342 2 9

39 2379 206 238 3 8

40 12 h 3138 186 314 3 8

41 1834 208 183 4 7

42 1560 211 156 5 6

43 24 h 2881 196 288 3 8

44 2204 208 220 4 7

45 1858 209 186 4 7


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion



12 h 12 h 12 h 12 h 25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

24 h 24 h 24 h 24 h 13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959


12 h 12 h 12 h 12 h 76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

24 h 24 h 24 h 24 h 78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665


12 h 12 h 12 h 12 h 66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

24 h 24 h 24 h 24 h 98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529


12 h 12 h 12 h 12 h 18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

24 h 24 h 24 h 24 h 48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991


12 h 12 h 12 h 12 h 137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

24h 24h 24h 24h 12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

226319271451 226319271451 188340381833 188340381833 738673147314 738673147314 1431344305739 1431344305739

32576573983 32576573983 33257839919 33257839919 35441232594 35441232594 66388345401 66388345401

13579208481 13579208481 24462095444 24462095444 12108228094 12108228094 18393683674 18393683674

11988964165 11988964165 12967268992 12967268992 07848466472 07848466472 13722458287 13722458287

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
NFkB genes (6h)

Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
100
100
100
100
100
100
100
100
100
100
100
100
1184911292873
42457060582
1043246428844
251503011717
675582299184
337905349034
616378320146
115343368286
47947993562
562826404461
548012911621
581135991695
1037790014802
295939942548
982084932456
156848314092
656644416767
20017142486
883157652966
72380508138
239629499071
240262650446
272994269647
166938074011
574249124853
169989137367
555154778548
65775511458
350689088998
193282389314
763920452803
52211813524
143502566696
158013625919
207094447769
54400087544
96360334555
195937197575
947294167043
210335597586
424648599422
3317708345
827268018455
221344262057
126073015371
23600471615
226127696746
97248127416

protocol

BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































Raw data BIRC3















Y-Intercept 227643

R^2 0999058






























































































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 2h 2379 2 238 3 8

2 2186 208 219 4 7

3 2741 199 274 3 8

4 12 h 2582 204 258 3 8

5 2450 201 245 3 8

6 1659 209 166 5 6

7 24 h 2005 21 201 4 7

8 1475 215 148 5 6

9 2135 204 214 4 7

10 64290694-AAA 10 mpk QD 2h 2747 199 275 3 8

11 2393 204 239 3 8

12 2211 206 221 4 7

13 12 h 2580 199 258 3 8

14 2985 196 299 3 8

15 2096 207 210 4 7

16 24h 1578 213 158 5 6

17 2483 196 248 3 8

18 2849 198 285 3 8

19 64290694-AAA 20 mpk QD 2h 2074 208 207 4 7

20 2668 203 267 3 8

21 2733 193 273 3 8

22 12 h 2299 205 230 3 8

23 2566 206 257 3 8

24 1237 217 124 6 5

25 24h 2236 202 224 4 7

26 2146 209 215 4 7

27 1489 21 149 5 6

28 64290694-AAA 40 mpk QD 2h 1338 212 134 6 5

29 1504 21 150 5 6

30 3203 188 320 2 9

31 12 h 2374 206 237 3 8

32 2557 204 256 3 8

33 2670 187 267 3 8

34 24h 2598 202 260 3 8

35 2485 205 249 3 8

36 2612 203 261 3 8

37 64290694-AAA 120 mpk QD 2h 1030 106 103 8 3

38 3419 146 342 2 9

39 2379 206 238 3 8

40 12 h 3138 186 314 3 8

41 1834 208 183 4 7

42 1560 211 156 5 6

43 24 h 2881 196 288 3 8

44 2204 208 220 4 7

45 1858 209 186 4 7


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion



12 h 12 h 12 h 12 h 25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

24 h 24 h 24 h 24 h 13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959


12 h 12 h 12 h 12 h 76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

24 h 24 h 24 h 24 h 78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665


12 h 12 h 12 h 12 h 66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

24 h 24 h 24 h 24 h 98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529


12 h 12 h 12 h 12 h 18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

24 h 24 h 24 h 24 h 48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991


12 h 12 h 12 h 12 h 137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

24h 24h 24h 24h 12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

226319271451 226319271451 188340381833 188340381833 738673147314 738673147314 1431344305739 1431344305739

32576573983 32576573983 33257839919 33257839919 35441232594 35441232594 66388345401 66388345401

13579208481 13579208481 24462095444 24462095444 12108228094 12108228094 18393683674 18393683674

11988964165 11988964165 12967268992 12967268992 07848466472 07848466472 13722458287 13722458287

Chart1

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)
100
100
100
100
100
100
100
100
100
100
100
100
1184911292873
42457060582
1043246428844
251503011717
675582299184
337905349034
616378320146
115343368286
47947993562
562826404461
548012911621
581135991695
1037790014802
295939942548
982084932456
156848314092
656644416767
20017142486
883157652966
72380508138
239629499071
240262650446
272994269647
166938074011
574249124853
169989137367
555154778548
65775511458
350689088998
193282389314
763920452803
52211813524
143502566696
158013625919
207094447769
54400087544
96360334555
195937197575
947294167043
210335597586
424648599422
3317708345
827268018455
221344262057
126073015371
23600471615
226127696746
97248127416

protocol

BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































Raw data BIRC3















Y-Intercept 227643

R^2 0999058






























































































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 2h 2379 2 238 3 8

2 2186 208 219 4 7

3 2741 199 274 3 8

4 12 h 2582 204 258 3 8

5 2450 201 245 3 8

6 1659 209 166 5 6

7 24 h 2005 21 201 4 7

8 1475 215 148 5 6

9 2135 204 214 4 7

10 64290694-AAA 10 mpk QD 2h 2747 199 275 3 8

11 2393 204 239 3 8

12 2211 206 221 4 7

13 12 h 2580 199 258 3 8

14 2985 196 299 3 8

15 2096 207 210 4 7

16 24h 1578 213 158 5 6

17 2483 196 248 3 8

18 2849 198 285 3 8

19 64290694-AAA 20 mpk QD 2h 2074 208 207 4 7

20 2668 203 267 3 8

21 2733 193 273 3 8

22 12 h 2299 205 230 3 8

23 2566 206 257 3 8

24 1237 217 124 6 5

25 24h 2236 202 224 4 7

26 2146 209 215 4 7

27 1489 21 149 5 6

28 64290694-AAA 40 mpk QD 2h 1338 212 134 6 5

29 1504 21 150 5 6

30 3203 188 320 2 9

31 12 h 2374 206 237 3 8

32 2557 204 256 3 8

33 2670 187 267 3 8

34 24h 2598 202 260 3 8

35 2485 205 249 3 8

36 2612 203 261 3 8

37 64290694-AAA 120 mpk QD 2h 1030 106 103 8 3

38 3419 146 342 2 9

39 2379 206 238 3 8

40 12 h 3138 186 314 3 8

41 1834 208 183 4 7

42 1560 211 156 5 6

43 24 h 2881 196 288 3 8

44 2204 208 220 4 7

45 1858 209 186 4 7


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion



12 h 12 h 12 h 12 h 25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

24 h 24 h 24 h 24 h 13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959


12 h 12 h 12 h 12 h 76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

24 h 24 h 24 h 24 h 78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665


12 h 12 h 12 h 12 h 66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

24 h 24 h 24 h 24 h 98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529


12 h 12 h 12 h 12 h 18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

24 h 24 h 24 h 24 h 48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991


12 h 12 h 12 h 12 h 137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

24h 24h 24h 24h 12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

protocol

BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































Raw data BIRC3















Y-Intercept 227643

R^2 0999058






























































































1 Aim




2 Products









primersetsRTQ PCR





3 Tumorsamples


4 RTQ PCR



Pre preparation


- tab off fresh N2











Repeat if necessary




42 RNA preparation



- vortex


- vortex













- DNAse treatment























- incubate 2 min








42 cDNA synthesis





DEPC-water x microl

11 microl

110 verdunning


1 JJN-3 vehicle (PEG400) 2h 2379 2 238 3 8

2 2186 208 219 4 7

3 2741 199 274 3 8

4 12 h 2582 204 258 3 8

5 2450 201 245 3 8

6 1659 209 166 5 6

7 24 h 2005 21 201 4 7

8 1475 215 148 5 6

9 2135 204 214 4 7

10 64290694-AAA 10 mpk QD 2h 2747 199 275 3 8

11 2393 204 239 3 8

12 2211 206 221 4 7

13 12 h 2580 199 258 3 8

14 2985 196 299 3 8

15 2096 207 210 4 7

16 24h 1578 213 158 5 6

17 2483 196 248 3 8

18 2849 198 285 3 8

19 64290694-AAA 20 mpk QD 2h 2074 208 207 4 7

20 2668 203 267 3 8

21 2733 193 273 3 8

22 12 h 2299 205 230 3 8

23 2566 206 257 3 8

24 1237 217 124 6 5

25 24h 2236 202 224 4 7

26 2146 209 215 4 7

27 1489 21 149 5 6

28 64290694-AAA 40 mpk QD 2h 1338 212 134 6 5

29 1504 21 150 5 6

30 3203 188 320 2 9

31 12 h 2374 206 237 3 8

32 2557 204 256 3 8

33 2670 187 267 3 8

34 24h 2598 202 260 3 8

35 2485 205 249 3 8

36 2612 203 261 3 8

37 64290694-AAA 120 mpk QD 2h 1030 106 103 8 3

38 3419 146 342 2 9

39 2379 206 238 3 8

40 12 h 3138 186 314 3 8

41 1834 208 183 4 7

42 1560 211 156 5 6

43 24 h 2881 196 288 3 8

44 2204 208 220 4 7

45 1858 209 186 4 7


per sample









per sample















Mastermix per well









BIRC-3 Hs00154109_m1


NFKB2 Hs00174517_m1


6 Conclusion


BIRC3


TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































Raw data BIRC3















Y-Intercept 227643

R^2 0999058





























































































TNFAIP3


GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































Raw data TNFAIP3















Y-Intercept 2382267

R^2 0995334





























































































GusB

ampCLJ160012 GusB

NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































Raw data GusB














Y-Intercept 2506271

R^2 099619





























































































NFKB2

ampCLJ160012 NFKB2

RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































Raw data NFKB2















R^2 099819493





























































































RelB


GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































Raw data RelB














R^2 099032915





























































































GusB_2

ampCLJ160012 GusB_2

summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

Raw data NFKBIE














R^2 098650163





























































































summary run 1


summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0905 1267 1894 0478 0669 106 95 0449 0706

2 1188 2054 2508 0474 0819 105 116

3 0999 1589 2517 0397 0631 88 89

4 12 h 1302 1446 1929 0675 0750 125 93 0541 0804

5 1024 1880 2592 0395 0725 73 90

6 0828 1401 1497 0553 0936 102 116

7 24 h 1736 1229 1325 1310 0928 108 130 1210 0715

8 1232 0684 0947 1301 0723 108 101

9 14589 7078 14309 1020 0495 84 69

10 64290694-AAA 10 mpk QD 6 h 4428 1915 7290 0607 0263 135 37

11 2908 1597 4968 0585 0321 130 45

12 1065 0831 2632 0405 0316 90 45

13 12 h 8370 5581 22894 0366 0244 68 30

14 10003 7031 30851 0324 0228 60 28

15 2726 2298 6700 0407 0343 75 43

16 24 h 3054 1841 6185 0494 0298 41 42

17 4849 3042 7117 0681 0427 56 60

18 3397 2895 6005 0566 0482 47 67

19 64290694-AAA 20 mpk QD 6 h 1886 0941 4836 0390 0195 87 28

20 3269 1364 7602 0430 0179 96 25

21 4187 1830 7228 0579 0253 129 36

22 12 h 0621 0242 1623 0382 0149 71 19

23 3355 1796 10675 0314 0168 58 21

24 0897 0401 2428 0369 0165 68 21

25 24 h 1001 0530 5999 0167 0088 14 12

26 3148 2038 7772 0405 0262 33 37

27 1726 0954 5790 0298 0165 25 23

28 64290694-AAA 40 mpk QD 6 h 0404 0207 1758 0230 0118 51 17

29 1003 0570 3903 0257 0146 57 21

30 4849 1632 16890 0287 0097 64 14

31 12 h 0567 0450 2977 0190 0151 35 19

32 0415 0345 2082 0199 0166 37 21

33 0471 0390 2620 0180 0149 33 19

34 24 h 0377 0238 2067 0182 0115 15 16

35 0628 0439 5643 0111 0078 9 11

36 0689 0443 3031 0227 0146 19 20

37 64290694-AAA 120 mpk QD 6 h 1908 0420 3644 0524 0115 117 16

38 0486 0141 1393 0349 0101 78 14

39 1529 0712 3582 0427 0199 95 28

40 12 h 1026 1489 7142 0144 0209 27 26

41 0376 0523 1388 0271 0377 50 47

42 0791 0617 2879 0275 0214 51 27

43 24 h 1066 1170 6310 0169 0185 14 26

44 0258 0288 1870 0138 0154 11 22

45 0369 0408 2446 0151 0167 12 23

3 Graph

stdev


vehicle 6 h 100 100 10 14

12 h 100 100 26 14

24 h 100 100 14 30

64290694-AAA 10 mpk QD 6 h 118 42 25 5

12 h 68 34 8 8

24 h 48 56 8 13

64290694-AAA 20 mpk QD 6 h 104 30 22 6

12 h 66 20 7 1

24 h 24 24 10 12

64290694-AAA 40 mpk QD 6 h 57 17 6 4

12 h 35 19 2 1

24 h 14 16 5 5

64290694-AAA 120 mpk QD 6 h 96 20 20 7

12 h 42 33 14 12

24h 13 24 1 2

summary run 1

BIRC3
TNFAIP3

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

101424440884 101424440884 140724097571 140724097571

25951707537 25951707537 143069422685 143069422685

13640263492 13640263492 302835030986 302835030986

247537439545 247537439545 45765131652 45765131652

76335628474 76335628474 77608533122 77608533122

78236096789 78236096789 132567869231 132567869231

222008380118 222008380118 55128685954 55128685954

66814604595 66814604595 1273436585 1273436585

98548565422 98548565422 121858432931 121858432931

6349531223 6349531223 35091190072 35091190072

18105372819 18105372819 11296909517 11296909517

48268731516 48268731516 47997749063 47997749063

195092246444 195092246444 74593942995 74593942995

137891846527 137891846527 119208540842 119208540842

12750574812 12750574812 22015498037 22015498037

summary run 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

1 Raw data



1 vehicle (PEG400) 6 h 0416 0917 0770 0540 1191 83 101 0653 1174

2 0721 1280 1063 0679 1204 104 103

3 0620 0945 0839 0739 1126 113 96

4 12 h 0523 0852 0916 0571 0931 98 101 0585 0917

5 0660 1007 1198 0551 0841 94 92

6 0402 0622 0635 0632 0980 108 107

7 24 h 1114 0728 0699 1594 1041 118 142 1349 0731

8 0732 0354 0412 1779 0859 132 118

9 4766 2064 7073 0674 0292 50 40

10 64290694-AAA 10 mpk QD 6 h 2356 0965 3265 0722 0295 111 25

11 1465 0745 2275 0644 0328 99 28

12 0642 0249 0948 0677 0263 104 22

13 12 h 3752 0861 10154 0370 0085 63 9

14 4251 1020 15556 0273 0066 47 7

15 1389 0529 3165 0439 0167 75 18

16 24 h 1409 0899 2726 0517 0330 38 45

17 2589 1482 3100 0835 0478 62 65

18 2687 1447 3104 0866 0466 64 64

19 64290694-AAA 20 mpk QD 6 h 1414 0430 2969 0476 0145 73 12

20 1718 0554 2849 0603 0195 92 17

21 2994 0755 3546 0844 0213 129 18

22 12 h 0589 0077 0800 0736 0096 126 11

23 1661 0335 8707 0191 0038 33 4

24 0803 0083 1290 0622 0064 106 7

25 24 h 0482 0114 2407 0200 0047 15 6

26 1566 0700 3227 0485 0217 36 30

27 1176 0286 2806 0419 0102 31 14

28 64290694-AAA 40 mpk QD 6 h 0362 0086 0829 0436 0104 67 9

29 0881 0198 2641 0334 0075 51 6

30 1842 0305 5807 0317 0053 49 4

31 12 h 0854 0100 1592 0536 0063 92 7

32 0462 0039 0966 0478 0040 82 4

33 0472 0058 1444 0327 0040 56 4

34 24 h 0402 0056 1212 0332 0046 25 6

35 0497 0076 2708 0184 0028 14 4

36 0492 0068 1525 0323 0045 24 6

37 64290694-AAA 120 mpk QD 6 h 2141 0578 2407 0889 0240 136 20

38 0475 0159 0995 0477 0160 73 14

39 1010 0704 2068 0488 0341 75 29

40 12 h 1186 0578 3413 0347 0169 59 18

41 0569 0229 0778 0731 0294 125 32

42 0628 0246 1682 0373 0146 64 16

43 24 h 0665 0161 2938 0226 0055 17 8

44 0360 0077 0933 0386 0082 29 11

45 0383 0096 1263 0303 0076 22 10

3 Graph

stdev


vehicle 6 h 100 100 16 4

12 h 100 100 7 8

24 h 100 100 44 53

64290694-AAA 10 mpk QD 6 h 104 25 6 3

12 h 62 12 14 6

24 h 55 58 14 11

64290694-AAA 20 mpk QD 6 h 98 16 29 3

12 h 88 7 49 3

24 h 27 17 11 12

64290694-AAA 40 mpk QD 6 h 56 7 10 2

12 h 76 5 18 1

24 h 21 5 6 1

64290694-AAA 120 mpk QD 6 h 95 21 36 8

12 h 83 22 37 9

24h 23 10 6 2

summary run 2

NFKB2
RelB

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

156046369579 156046369579 35752747 35752747

72323084851 72323084851 76806376302 76806376302

438728626462 438728626462 534869968959 534869968959

59679940164 59679940164 27686893639 27686893639

142016957804 142016957804 58771706119 58771706119

143171623674 143171623674 112667394665 112667394665

286437877673 286437877673 30137517793 30137517793

492160064437 492160064437 31640430444 31640430444

11068446609 11068446609 118371935529 118371935529

9878134817 9878134817 22026591807 22026591807

184834140604 184834140604 14137146939 14137146939

61571099954 61571099954 13858667991 13858667991

359609591073 359609591073 77250566828 77250566828

366446937303 366446937303 8654863017 8654863017

59076290074 59076290074 19597914187 19597914187

Summary

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

Summary

stdev


vehicle 6 h 100 100 100 100 10 14 16 4

12 h 100 100 100 100 26 14 7 8

24 h 100 100 100 100 14 30 44 53

10 mpk QD 6 h 118 42 104 25 25 5 6 3

12 h 68 34 62 12 8 8 14 6

24 h 48 56 55 58 8 13 14 11

20 mpk QD 6 h 104 30 98 16 22 6 29 3

12 h 66 20 88 7 7 1 49 3

24 h 24 24 27 17 10 12 11 12

40 mpk QD 6 h 57 17 56 7 6 4 10 2

12 h 35 19 76 5 2 1 18 1

24 h 14 16 21 5 5 5 6 1

120 mpk QD 6 h 96 20 95 21 20 7 36 8

12 h 42 33 83 22 14 12 37 9

24h 13 24 23 10 1 2 6 2

Summary

BIRC3
TNFAIP3
NFKB2
RelB

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

BIRC3
TNFAIP3
NFKB2
RelB
TRC694
Q-PCR ( of vehicle)

Slide Number 1

101424440884 101424440884 140724097571 140724097571 156046369579 156046369579 35752747 35752747

25951707537 25951707537 143069422685 143069422685 72323084851 72323084851 76806376302 76806376302

13640263492 13640263492 302835030986 302835030986 438728626462 438728626462 534869968959 534869968959

247537439545 247537439545 45765131652 45765131652 59679940164 59679940164 27686893639 27686893639

76335628474 76335628474 77608533122 77608533122 142016957804 142016957804 58771706119 58771706119

78236096789 78236096789 132567869231 132567869231 143171623674 143171623674 112667394665 112667394665

222008380118 222008380118 55128685954 55128685954 286437877673 286437877673 30137517793 30137517793

66814604595 66814604595 1273436585 1273436585 492160064437 492160064437 31640430444 31640430444

98548565422 98548565422 121858432931 121858432931 11068446609 11068446609 118371935529 118371935529

6349531223 6349531223 35091190072 35091190072 9878134817 9878134817 22026591807 22026591807

18105372819 18105372819 11296909517 11296909517 184834140604 184834140604 14137146939 14137146939

48268731516 48268731516 47997749063 47997749063 61571099954 61571099954 13858667991 13858667991

195092246444 195092246444 74593942995 74593942995 359609591073 359609591073 77250566828 77250566828

137891846527 137891846527 119208540842 119208540842 366446937303 366446937303 8654863017 8654863017

12750574812 12750574812 22015498037 22015498037 59076290074 59076290074 19597914187 19597914187

101424440884	101424440884	140724097571	140724097571	156046369579	156046369579	35752747	35752747

25951707537	25951707537	143069422685	143069422685	72323084851	72323084851	76806376302	76806376302

13640263492	13640263492	302835030986	302835030986	438728626462	438728626462	534869968959	534869968959

247537439545	247537439545	45765131652	45765131652	59679940164	59679940164	27686893639	27686893639

76335628474	76335628474	77608533122	77608533122	142016957804	142016957804	58771706119	58771706119

78236096789	78236096789	132567869231	132567869231	143171623674	143171623674	112667394665	112667394665

222008380118	222008380118	55128685954	55128685954	286437877673	286437877673	30137517793	30137517793

66814604595	66814604595	1273436585	1273436585	492160064437	492160064437	31640430444	31640430444

98548565422	98548565422	121858432931	121858432931	11068446609	11068446609	118371935529	118371935529

6349531223	6349531223	35091190072	35091190072	9878134817	9878134817	22026591807	22026591807

18105372819	18105372819	11296909517	11296909517	184834140604	184834140604	14137146939	14137146939

48268731516	48268731516	47997749063	47997749063	61571099954	61571099954	13858667991	13858667991

195092246444	195092246444	74593942995	74593942995	359609591073	359609591073	77250566828	77250566828

137891846527	137891846527	119208540842	119208540842	366446937303	366446937303	8654863017	8654863017

12750574812	12750574812	22015498037	22015498037	59076290074	59076290074	19597914187	19597914187

	Summary

						stdev

		BIRC3		TNFAIP3		NFKB2		RelB		BIRC3		TNFAIP3		NFKB2		RelB

vehicle		6 h		100		100		100		100		10		14		16		4

	12 h		100		100		100		100		26		14		7		8

	24 h		100		100		100		100		14		30		44		53

10 mpk QD		6 h		118		42		104		25		25		5		6		3

	12 h		68		34		62		12		8		8		14		6

	24 h		48		56		55		58		8		13		14		11

20 mpk QD		6 h		104		30		98		16		22		6		29		3

	12 h		66		20		88		7		7		1		49		3

	24 h		24		24		27		17		10		12		11		12

40 mpk QD		6 h		57		17		56		7		6		4		10		2

	12 h		35		19		76		5		2		1		18		1

	24 h		14		16		21		5		5		5		6		1

120 mpk QD		6 h		96		20		95		21		20		7		36		8

	12 h		42		33		83		22		14		12		37		9

	24h		13		24		23		10		1		2		6		2

			Summary

1 Raw data

				QTY Mean				Ratio			control			Mean Ratio Vehicle

				NFKB2		RelB		GusB		NFKB2GusB		RelBGusB		NFKB2		RelB		NFKB2		RelB

1		vehicle (PEG400)		6 h			0416		0917		0770		0540		1191		83		101		0653		1174

2					0721		1280		1063		0679		1204		104		103

3					0620		0945		0839		0739		1126		113		96

4			12 h			0523		0852		0916		0571		0931		98		101		0585		0917

5					0660		1007		1198		0551		0841		94		92

6					0402		0622		0635		0632		0980		108		107

7			24 h			1114		0728		0699		1594		1041		118		142		1349		0731

8					0732		0354		0412		1779		0859		132		118

9					4766		2064		7073		0674		0292		50		40

10		64290694-AAA 10 mpk QD		6 h			2356		0965		3265		0722		0295		111		25

11					1465		0745		2275		0644		0328		99		28

12					0642		0249		0948		0677		0263		104		22

13			12 h			3752		0861		10154		0370		0085		63		9

14					4251		1020		15556		0273		0066		47		7

15					1389		0529		3165		0439		0167		75		18

16			24 h			1409		0899		2726		0517		0330		38		45

17					2589		1482		3100		0835		0478		62		65

18					2687		1447		3104		0866		0466		64		64

19		64290694-AAA 20 mpk QD		6 h			1414		0430		2969		0476		0145		73		12

20					1718		0554		2849		0603		0195		92		17

21					2994		0755		3546		0844		0213		129		18

22			12 h			0589		0077		0800		0736		0096		126		11

23					1661		0335		8707		0191		0038		33		4

24					0803		0083		1290		0622		0064		106		7

25			24 h			0482		0114		2407		0200		0047		15		6

26					1566		0700		3227		0485		0217		36		30

27					1176		0286		2806		0419		0102		31		14

28		64290694-AAA 40 mpk QD		6 h			0362		0086		0829		0436		0104		67		9

29					0881		0198		2641		0334		0075		51		6

30					1842		0305		5807		0317		0053		49		4

31			12 h			0854		0100		1592		0536		0063		92		7

32					0462		0039		0966		0478		0040		82		4

33					0472		0058		1444		0327		0040		56		4

34			24 h			0402		0056		1212		0332		0046		25		6

35					0497		0076		2708		0184		0028		14		4

36					0492		0068		1525		0323		0045		24		6

37		64290694-AAA 120 mpk QD		6 h			2141		0578		2407		0889		0240		136		20

38					0475		0159		0995		0477		0160		73		14

39					1010		0704		2068		0488		0341		75		29

40			12 h			1186		0578		3413		0347		0169		59		18

41					0569		0229		0778		0731		0294		125		32

42					0628		0246		1682		0373		0146		64		16

43			24 h			0665		0161		2938		0226		0055		17		8

44					0360		0077		0933		0386		0082		29		11

45					0383		0096		1263		0303		0076		22		10

3 Graph

						stdev

				NFKB2		RelB		NFKB2		RelB

	vehicle		6 h			100		100		16		4

		12 h			100		100		7		8

		24 h			100		100		44		53

	64290694-AAA 10 mpk QD		6 h			104		25		6		3

		12 h			62		12		14		6

		24 h			55		58		14		11

	64290694-AAA 20 mpk QD		6 h			98		16		29		3

		12 h			88		7		49		3

		24 h			27		17		11		12

	64290694-AAA 40 mpk QD		6 h			56		7		10		2

		12 h			76		5		18		1

		24 h			21		5		6		1

	64290694-AAA 120 mpk QD		6 h			95		21		36		8

		12 h			83		22		37		9

		24h			23		10		6		2

LJ160012 RTQ PCR JJN-3 PD study

1 Aim

In vivo PD study to test the anti-tumour effect of 64290694-AAA in a subcutaneous JJN-3 xenograft model

Take samples of the mice at 212 and 24 h after treatment

Is there an effect on the NIK-driven genes (BIRC-3 NFKB2 and TNFAIP3)

2 Products

Qiazol Lysis reagent				Qiagen				79306

Rneasy mini kit				Qiagen				74106

Qiaschreddar				Qiagen				79656

QPCR Core kit				Eurogentec				RT-QP10-05NU

oligodT (12-18)				Invitrogen				SKU 18418-012				25 microg

superscript II reverse transcriptase				invitrogen				SKU 18064-071				4x10000 U

10 mM dNTP Mix				invitrogen				SKU 18427-088				1 ml

DNAse				Qiagen				QIAG79254

primersetsRTQ PCR

	BIRC-3			Applied Biosystems				Hs00154109_m1

	TNFAIP3			Applied Biosystems				Hs00234713_m1

	NFKB2			Applied Biosystems				Hs00174517_m1

	GusB			Applied Biosystems				Hs00939627_m1

3 Tumorsamples

see protocol OIV154583

4 RTQ PCR

41 Pulverize the tumor samples

httpcovarisinccomcryoprephtm

Pre preparation

- Store the tumor samples at dry ice (plus extra eppendorf 15 ml vials)

- tab off fresh N2

- store the black massive block also in dry ice and let it cool down

- put already in the black massive block the tissueTubes and the glass vials to already cool down

- put the Cryoprep system on

If everything is cold enough then we can start using teh Cryoprep system

Take teh tube with the tumor inside and freeze it in the N2 for several seconds In the mean time with your other hand the glass vials should be screwed onto the tissuetubes

and also frozen in the N2 for several seconds unscrew again the tissuetube from the glass vial Then open the tube and try to get the tumor into the TissueTube

If the tumor is settled in the TissueTube again screw the glass vial unto the TissueTube and put the TissueTube into the liquid N2 for at least 30 s

Then put the whole construction in the machine With the glass vial above and underneath the TissueTube Make sure that the glassvial is not completely tightent to the

TissueTube so that air can escape during the Pulverize

Pulverize at stand 1 for small tumors or stand 2 for bigger tumors (in case of bone kidney or skin tisue use an higher pulverize stand)

Repeat if necessary

Freeze again the TissueTube1 in the liquid N2 and then put everything to on side

Cut at the other side a tip of the TissueTube1 with a pair of scissors and shake everything back into the original sample tube or into a new tube

Freeze the sample back in the liquid N2 and put the samples back into -80degC

42 RNA preparation

- store samples at -80degC

- add 700 microl QIAzol Lysis reagent

- vortex

- add 140 microl chloroform

- vortex

- centrifuge at 6000 g for 15 min at 4degC

	after centrifugation the sample seperates into 3 phases upper colorless aqueous phase containing RNA a white interphase and a lower red organic phase

	For tissues with an especially high fat content an additional clear phase may be visible below the red organic phase

	The volume of the aqueous phase shpuld be approximately 400 microl

- transfer all of the upper aqueous phases to a new eppendorf tube

- add 400 microl 70 EtOH

- mix by pipetting up and down

- put the mix in the Rneasy columns

- spin 15s at max speed at RT

- discard the flow-through

- add 350 microl RW1 buffer to the columns

- spin 15s at high speed

- DNAse treatment

	add 10 microl DNAse stock to 70 microl RDD buffer

	mix gently by inverting

	pipet 80 microl of the DNAse incubation mix directly onto the Rneasy membrane in each well of the plate

	seal with Airpore Tape sheet

	place at RT for 15 min

	remove the Airpore tape

- add 350 microl of buffer RW1 to each column (inhibition of DNAse)

- wait 5 min at RT (to efficiently remove the DNAse digestion)

- centrifuge 15s at max speed at RT

- discard the flow-through

- add 500 microl RPE buffer onto the columns

	worksolution add 4 volumes EtOH to the RPE buffer

- spin for 15s at max speed at RT

- discard the flow-through

- add another 500 microl RPE buffer onto the columns

- spin for 15s at max speed at RT

- discard the flow-through

- centrifuge 5 min at max speed to dry the column completely

	its important to dry the Rneasy membrane since residual EtOH may interfere with subsequent reactions

- transfer column to a 15 ml collection tube

	Do not carry over any flow-trough

- add 30 microl RNAse free water onto the middle of the membrane

- incubate 2 min

- centrifuge for 1 min at max speed to elute the RNA

- repeat the elution with 30 microl RNAse free water to elute the RNA

- measure the concentration of all RNA samples using the Nanodrop8000

	if the concentration is not high enough speedvac all the samples during 15 min at 45degC

	check the quality of the RNA if 260280 is higher than 2 the quality is OK

- dilute the RNA to 1 microg or somewhere between 400-600 ng

	dilute RNA in a volume of 50 microl to equal concentrations

42 cDNA synthesis

- the cDNA synthesis is performed in a PCR machine

	- add the following components to a nuclease-free eppendorf tube

	- dilute RNA to x ng in DEPC-water

		x microg RNA			x microl

		DEPC-water			x microl

				11 microl

									110 verdunning

				ngmicrol		260280		110		800 ng		RNAse free water		800 ng		add RNAse free water to 11 microl

1		JJN-3		vehicle (PEG400)		2h		2379		2		238				3		8

2					2186		208		219				4		7

3					2741		199		274				3		8

4				12 h		2582		204		258				3		8

5					2450		201		245				3		8

6					1659		209		166				5		6

7				24 h		2005		21		201				4		7

8					1475		215		148				5		6

9					2135		204		214				4		7

10			64290694-AAA 10 mpk QD		2h		2747		199		275				3		8

11					2393		204		239				3		8

12					2211		206		221				4		7

13				12 h		2580		199		258				3		8

14					2985		196		299				3		8

15					2096		207		210				4		7

16				24h		1578		213		158				5		6

17					2483		196		248				3		8

18					2849		198		285				3		8

19			64290694-AAA 20 mpk QD		2h		2074		208		207				4		7

20					2668		203		267				3		8

21					2733		193		273				3		8

22				12 h		2299		205		230				3		8

23					2566		206		257				3		8

24					1237		217		124				6		5

25				24h		2236		202		224				4		7

26					2146		209		215				4		7

27					1489		21		149				5		6

28			64290694-AAA 40 mpk QD		2h		1338		212		134				6		5

29					1504		21		150				5		6

30					3203		188		320				2		9

31				12 h		2374		206		237				3		8

32					2557		204		256				3		8

33					2670		187		267				3		8

34				24h		2598		202		260				3		8

35					2485		205		249				3		8

36					2612		203		261				3		8

37			64290694-AAA 120 mpk QD		2h		1030		106		103				8		3

38					3419		146		342				2		9

39					2379		206		238				3		8

40				12 h		3138		186		314				3		8

41					1834		208		183				4		7

42					1560		211		156				5		6

43				24 h		2881		196		288				3		8

44					2204		208		220				4		7

45					1858		209		186				4		7

		- prepare mix A of the following components in a nuclease-free eppendorf tube

					per sample

			Random Primer (30microgmicrol)			0167 microl

			Oligo dT (05 microgml)			1 microl

			DEPC-water			233 microl

		- add 35 microl from mix A to the diluted RNA (total volume = 145 microl)

		- heat to 70degC for 5 min

		- quick chill on ice

		- collect the contents of the tube by brief centrifugation

		- prepare mix B of the following components in a nuclease-free eppendorf tube

					per sample

			dNTP (20 mM)			05 microl

			M-MLV RT 5x buffer			4 microl

			M-MLV RT RNAse Point mutant			05 microl

			DEPC-water			05 microl

		- add 55 microl from mix B to the RNA

		- collect the contents of the tube by brief centrifugation

		- incubate at 20degC for 10 min

		- incubate at 40degC for 50 min

		- inactivate the reaction by heating at 70degC for 15 min

		- store the cDNA at -20degC

43 RTQ PCR using CAS-1200 Corbett Robotics

	According SOP Functional Genomics RTQ PCR set-up CAS1200 and Biomek 3000 robot

		Functional Genomics RTQ PCR ABI7900HT

	per 384-well RTQplate 128 samples can be tested in triplicate (2 times 23 of a 96-well plate)

	Mastermix per well

	RNAse free water				1815 microl

	10x PCR buffer A (Eurogentec)				3 microl

	MgCl2 (Eurogentec)				3 microl

	dNTP mix 5 mM (Eurogentec)				12 microl

	taqman gene expression assays				15 microl

	HotGoldStar enzyme				015 microl

				27 microl mix + 3 microl cDNA sample

	taqman gene expression assays (Applied Biosystems)

	BIRC-3				Hs00154109_m1

	TNFAIP3				Hs00234713_m1

	NFKB2				Hs00174517_m1

	housekeeping genes GUS-b				Hs99999908_m1

6 Conclusion

There is a nice respression of most of the genes and the respression is the highest 24h after the last dose

6 hvehicle	6 hvehicle	6 hvehicle	6 hvehicle	101424440884	101424440884	140724097571	140724097571	156046369579	156046369579	35752747	35752747

12 h	12 h	12 h	12 h	25951707537	25951707537	143069422685	143069422685	72323084851	72323084851	76806376302	76806376302

24 h	24 h	24 h	24 h	13640263492	13640263492	302835030986	302835030986	438728626462	438728626462	534869968959	534869968959

6 h 10 mpk QD	6 h 10 mpk QD	6 h 10 mpk QD	6 h 10 mpk QD	247537439545	247537439545	45765131652	45765131652	59679940164	59679940164	27686893639	27686893639

12 h	12 h	12 h	12 h	76335628474	76335628474	77608533122	77608533122	142016957804	142016957804	58771706119	58771706119

24 h	24 h	24 h	24 h	78236096789	78236096789	132567869231	132567869231	143171623674	143171623674	112667394665	112667394665

6 h20 mpk QD	6 h20 mpk QD	6 h20 mpk QD	6 h20 mpk QD	222008380118	222008380118	55128685954	55128685954	286437877673	286437877673	30137517793	30137517793

12 h	12 h	12 h	12 h	66814604595	66814604595	1273436585	1273436585	492160064437	492160064437	31640430444	31640430444

24 h	24 h	24 h	24 h	98548565422	98548565422	121858432931	121858432931	11068446609	11068446609	118371935529	118371935529

6 h 40 mpk QD	6 h 40 mpk QD	6 h 40 mpk QD	6 h 40 mpk QD	6349531223	6349531223	35091190072	35091190072	9878134817	9878134817	22026591807	22026591807

12 h	12 h	12 h	12 h	18105372819	18105372819	11296909517	11296909517	184834140604	184834140604	14137146939	14137146939

24 h	24 h	24 h	24 h	48268731516	48268731516	47997749063	47997749063	61571099954	61571099954	13858667991	13858667991

6 h 120 mpk QD	6 h 120 mpk QD	6 h 120 mpk QD	6 h 120 mpk QD	195092246444	195092246444	74593942995	74593942995	359609591073	359609591073	77250566828	77250566828

12 h	12 h	12 h	12 h	137891846527	137891846527	119208540842	119208540842	366446937303	366446937303	8654863017	8654863017

24h	24h	24h	24h	12750574812	12750574812	22015498037	22015498037	59076290074	59076290074	19597914187	19597914187

226319271451	226319271451	188340381833	188340381833	738673147314	738673147314	1431344305739	1431344305739

32576573983	32576573983	33257839919	33257839919	35441232594	35441232594	66388345401	66388345401

13579208481	13579208481	24462095444	24462095444	12108228094	12108228094	18393683674	18393683674

11988964165	11988964165	12967268992	12967268992	07848466472	07848466472	13722458287	13722458287

			Summary

1 Raw data							0345

				QTY Mean						Ratio					control					Mean Ratio Vehicle

				BIRC-3		TNFAIP3		NFKB2		RelB		GusB		BIRC3GusB		TNFAIP3GusB		NFKB2GusB		RelBGusB		BIRC3		TNFAIP3		NFKB2		RelB		BIRC3		TNFAIP3		NFKB2		RelB

1		vehicle (PEG400)		6 h			0097		0159		0345		0813		0114		0849		1395		3028		7138		135		127		237		384		0628		1101		1279	1858

3					2236		4062		4381		4036		3079		0726		1319		1423		1311		116		120		111		71

4					1278		1778		2634		3363		1967		0650		0904		1339		1710		103		82		105		92

5					4893		9615		8983		1197		10143		0482		0948		0886		0118		77		86		69		6

6					3092		6006		3151		3630		5571		0555		1078		0566		0652		88		98		44		35

7					5840		11179		5016		2555		11588		0504		0965		0433		0221		80		88		34		12

11		64290694-AAA 10 mpk QD				0314		0504		0543		1046		3039		0103		0166		0179		0344		16		15		14		19

12					0616		1839		0828		0454		13357		0046		0138		0062		0034		7		13		5		2

15					2320		3581		2163		0319		35091		0066		0102		0062		0009		11		9		5		0

16					0363		0874		0477		0500		4925		0074		0178		0097		0101		12		16		8		5

17					0582		1112		0700		0462		6157		0094		0181		0114		0075		15		16		9		4

18					0541		1533		0504		0323		7449		0073		0206		0068		0043		12		19		5		2

21		64290694-AAA 20 mpk QD				0136		0367		0178		0436		3806		0036		0096		0047		0115		6		9		4		6

22					0084		0309		0194		0136		3349		0025		0092		0058		0041		4		8		5		2

24					0116		0423		0223		0196		4718		0025		0090		0047		0042		4		8		4		2

25					0194		0709		0316		0388		5774		0034		0123		0055		0067		5		11		4		4

26					0127		0446		0237		0288		6879		0018		0065		0034		0042		3		6		3		2

27					0268		0894		0317		0303		19854		0013		0045		0016		0015		2		4		1		1

32		64290694-AAA 40 mpk QD				0173		0344		0238		0489		4757		0036		0072		0050		0103		6		7		4		6

33					0195		0446		0198		0310		4845		0040		0092		0041		0064		6		8		3		3

34					0311		0638		0312		0367		11920		0026		0053		0026		0031		4		5		2		2

35					0107		0363		0137		0315		4570		0023		0079		0030		0069		4		7		2		4

36					0134		0330		0144		0221		5601		0024		0059		0026		0039		4		5		2		2

37					0190		0406		0136		0271		5117		0037		0079		0027		0053		6		7		2		3

2 Mean

				control					Mean control					stdev

				BIRC3		TNFAIP3		NFKB2		RelB		BIRC3		TNFAIP3		NFKB2		RelB		BIRC3		TNFAIP3		NFKB2		RelB

1		vehicle (PEG400)		6 h			135		127		237		384		100		100		100		100		23		19		74		143

3					116		120		111		71

4					103		82		105		92

5					77		86		69		6

6					88		98		44		35

7					80		88		34		12

11		64290694-AAA 10 mpk QD				16		15		14		19		12		15		8		5		3		3		4		7

12					7		13		5		2

15					11		9		5		0

16					12		16		8		5

17					15		16		9		4

18					12		19		5		2

21		64290694-AAA 20 mpk QD				6		9		4		6		4		8		3		3		1		2		1		2

22					4		8		5		2

24					4		8		4		2

25					5		11		4		4

26					3		6		3		2

27					2		4		1		1

32		64290694-AAA 40 mpk QD				6		7		4		6		5		7		3		3		1		1		1		1

33					6		8		3		3

34					4		5		2		2

35					4		7		2		4

36					4		5		2		2

37					6		7		2		3

3 Graph

				Mean control					stdev

				BIRC3		TNFAIP3		NFKB2		RelB		BIRC3		TNFAIP3		NFKB2		Relb

	vehicle				100		100		100		100		23		19		74		143

	10 mpk QD				12		15		8		5		3		3		4		7

	20 mpk QD				4		8		3		3		1		2		1		2

	40 mpk QD				5		7		3		3		1		1		1		1

LJ150095 RTQ PCR JJN-3 efficacy study

1 Aim

In vivo efficacy study to test the anti-tumour effect of 64290694-AAA in a subcutaneous JJN-3 xenograft model

Take samples of the mice at 6h after treatment

Is there an effect on the NIK-driven genes (BIRC-3 NFKB2 and TNFAIP3)

2 Products

Qiazol Lysis reagent				Qiagen				79306

Rneasy mini kit				Qiagen				74106

Qiaschreddar				Qiagen				79656

QPCR Core kit				Eurogentec				RT-QP10-05NU

oligodT (12-18)				Invitrogen				SKU 18418-012				25 microg

superscript II reverse transcriptase				invitrogen				SKU 18064-071				4x10000 U

10 mM dNTP Mix				invitrogen				SKU 18427-088				1 ml

DNAse				Qiagen				QIAG79254

primersetsRTQ PCR

	BIRC-3			Applied Biosystems				Hs00154109_m1

	TNFAIP3			Applied Biosystems				Hs00234713_m1

	NFKB2			Applied Biosystems				Hs00174517_m1

	GusB			Applied Biosystems				Hs00939627_m1

3 Tumorsamples

see protocol OIV154567

4 RTQ PCR

41 Pulverize the tumor samples

httpcovarisinccomcryoprephtm

Pre preparation

- Store the tumor samples at dry ice (plus extra eppendorf 15 ml vials)

- tab off fresh N2

- store the black massive block also in dry ice and let it cool down

- put already in the black massive block the tissueTubes and the glass vials to already cool down

- put the Cryoprep system on

If everything is cold enough then we can start using teh Cryoprep system

Take teh tube with the tumor inside and freeze it in the N2 for several seconds In the mean time with your other hand the glass vials should be screwed onto the tissuetubes

and also frozen in the N2 for several seconds unscrew again the tissuetube from the glass vial Then open the tube and try to get the tumor into the TissueTube

If the tumor is settled in the TissueTube again screw the glass vial unto the TissueTube and put the TissueTube into the liquid N2 for at least 30 s

Then put the whole construction in the machine With the glass vial above and underneath the TissueTube Make sure that the glassvial is not completely tightent to the

TissueTube so that air can escape during the Pulverize

Pulverize at stand 1 for small tumors or stand 2 for bigger tumors (in case of bone kidney or skin tisue use an higher pulverize stand)

Repeat if necessary

Freeze again the TissueTube1 in the liquid N2 and then put everything to on side

Cut at the other side a tip of the TissueTube1 with a pair of scissors and shake everything back into the original sample tube or into a new tube

Freeze the sample back in the liquid N2 and put the samples back into -80degC

42 RNA preparation

- store samples at -80degC

- add 700 microl QIAzol Lysis reagent

- vortex

- add 140 microl chloroform

- vortex

- centrifuge at 6000 g for 15 min at 4degC

	after centrifugation the sample seperates into 3 phases upper colorless aqueous phase containing RNA a white interphase and a lower red organic phase

	For tissues with an especially high fat content an additional clear phase may be visible below the red organic phase

	The volume of the aqueous phase shpuld be approximately 400 microl

- transfer all of the upper aqueous phases to a new eppendorf tube

- add 400 microl 70 EtOH

- mix by pipetting up and down

- put the mix in the Rneasy columns

- spin 15s at max speed at RT

- discard the flow-through

- add 350 microl RW1 buffer to the columns

- spin 15s at high speed

- DNAse treatment

	add 10 microl DNAse stock to 70 microl RDD buffer

	mix gently by inverting

	pipet 80 microl of the DNAse incubation mix directly onto the Rneasy membrane in each well of the plate

	seal with Airpore Tape sheet

	place at RT for 15 min

	remove the Airpore tape

- add 350 microl of buffer RW1 to each column (inhibition of DNAse)

- wait 5 min at RT (to efficiently remove the DNAse digestion)

- centrifuge 15s at max speed at RT

- discard the flow-through

- add 500 microl RPE buffer onto the columns

	worksolution add 4 volumes EtOH to the RPE buffer

- spin for 15s at max speed at RT

- discard the flow-through

- add another 500 microl RPE buffer onto the columns

- spin for 15s at max speed at RT

- discard the flow-through

- centrifuge 5 min at max speed to dry the column completely

	its important to dry the Rneasy membrane since residual EtOH may interfere with subsequent reactions

- transfer column to a 15 ml collection tube

	Do not carry over any flow-trough

- add 30 microl RNAse free water onto the middle of the membrane

- incubate 2 min

- centrifuge for 1 min at max speed to elute the RNA

- repeat the elution with 30 microl RNAse free water to elute the RNA

- measure the concentration of all RNA samples using the Nanodrop8000

	if the concentration is not high enough speedvac all the samples during 15 min at 45degC

	check the quality of the RNA if 260280 is higher than 2 the quality is OK

- dilute the RNA to 1 microg or somewhere between 400-600 ng

	dilute RNA in a volume of 50 microl to equal concentrations

42 cDNA synthesis

- the cDNA synthesis is performed in a PCR machine

	- add the following components to a nuclease-free eppendorf tube

	- dilute RNA to x ng in DEPC-water

		x microg RNA			x microl

		DEPC-water			x microl

				11 microl

									110 verdunning

				ngmicrol		260280		110		800 ng		RNAse free water		800 ng		add RNAse free water to 11 microl

1		JJN-3		vehicle (PEG400)		6 h		4987		203		499				2	9

3					2189		217		219				4		7

4					1796		217		180				4		7

5					4665		206		466				2		9

6					2180		216		218				4		7

7					3386		215		339				2		9

11			64290694-AAA 10 mpk QD			3183		215		318				3		8

12					2569		217		257				3		8

15					3985		213		399				2		9

16					676		217		68				12		-1

17					4704		206		470				2		9

18					2644		217		264				3		8

21			64290694-AAA 20 mpk QD			1455		218		145				5		6

22					3482		215		348				2		9

24					902		217		90				9		2

25					3255		216		326				2		9

26					2316		217		232				3		8

27					3424		213		342				2		9

32			64290694-AAA 40 mpk QD			690		219		69				12		-1

33					1922		218		192				4		7

34					2207		216		221				4		7

35					2185		217		218				4		7

36					155		214		15		5		6

37					67		203		7		12		-1

		- prepare mix A of the following components in a nuclease-free eppendorf tube

					per sample

			Random Primer (30microgmicrol)			0167 microl

			Oligo dT (05 microgml)			1 microl

			DEPC-water			233 microl

		- add 35 microl from mix A to the diluted RNA (total volume = 145 microl)

		- heat to 70degC for 5 min

		- quick chill on ice

		- collect the contents of the tube by brief centrifugation

		- prepare mix B of the following components in a nuclease-free eppendorf tube

					per sample

			dNTP (20 mM)			05 microl

			M-MLV RT 5x buffer			4 microl

			M-MLV RT RNAse Point mutant			05 microl

			DEPC-water			05 microl

		- add 55 microl from mix B to the RNA

		- collect the contents of the tube by brief centrifugation

		- incubate at 20degC for 10 min

		- incubate at 40degC for 50 min

		- inactivate the reaction by heating at 70degC for 15 min

		- store the cDNA at -20degC

43 RTQ PCR using CAS-1200 Corbett Robotics

	According SOP Functional Genomics RTQ PCR set-up CAS1200 and Biomek 3000 robot

		Functional Genomics RTQ PCR ABI7900HT

	per 384-well RTQplate 128 samples can be tested in triplicate (2 times 23 of a 96-well plate)

	Mastermix per well

	RNAse free water				1815 microl

	10x PCR buffer A (Eurogentec)				3 microl

	MgCl2 (Eurogentec)				3 microl

	dNTP mix 5 mM (Eurogentec)				12 microl

	taqman gene expression assays				15 microl

	HotGoldStar enzyme				015 microl

				27 microl mix + 3 microl cDNA sample

	taqman gene expression assays (Applied Biosystems)

	BIRC-3				Hs00154109_m1

	TNFAIP3				Hs00234713_m1

	NFKB2				Hs00174517_m1

	housekeeping genes GUS-b				Hs99999908_m1

6 Conclusion

	JNJ-64290694-AAA (PEG400 solution) has a significant anti-tumor efficacy in the JJN-3 xenograft model

The effect on the genes is complet in line with what we expected

The efficacy and level of PD shutdown seems to be better with PEG400 dosing compared to

suspension dosing

# 4199 inhibition of nfκb-inducing kinase (nik ... · matthias versele1, lut janssen1, tamara...

Documents

Chart1

protocol

BIRC3

TNFAIP3

NFKB2

RelB

GusB

summary

summary

Chart1

protocol

BIRC3

TNFAIP3

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NFKB2

RelB

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summary run 1

summary run 1

summary run 2

summary run 2

Summary

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Chart1

protocol

BIRC3

TNFAIP3

NFKB2

RelB

GusB

summary

summary

Chart1

protocol

BIRC3

TNFAIP3

GusB

NFKB2

RelB

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summary run 1

summary run 1

summary run 2

summary run 2

Summary

Summary

protocol

BIRC3

TNFAIP3

NFKB2

RelB

GusB

summary

summary

Chart1

protocol

BIRC3

TNFAIP3

GusB

NFKB2

RelB

GusB_2

summary run 1

summary run 1

summary run 2

summary run 2

Summary

Summary

BIRC3

TNFAIP3

NFKB2

RelB

GusB

summary

summary

Chart1

protocol

BIRC3