mrna & microrna integrity - the key to success · mrna & microrna integrity - the key to...

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mRNA & microRNA integrity - the key to success

Michael W. PfafflChristiane Becker, Andrea Hammerle-Fickinger & Irmgard Riedmaier

Michael W. Pfaffl [email protected] – WeihenstephanTechnical University of Munich www.Gene-Quantification.infoWeihenstephaner Berg 385350 Freising-WeihenstephanGermany

qRT-PCR efficiency &overall reaction

performance

PCR inhibitors:Hemoglobin, Urea, Heparin

organic or phenolic compoundsGlycogen, Fatty acids, Ca2+

tissue matrix effects ( ? )laboratory items, powder, etc.

RNA / DNA degradation

unspecific PCR products

DNA concentration

sample degradation& tissue degradation

PCR enhancers:DMSO, Glycerol, BSA

Formamide, PEG, TMANO, TMAC etc.Special commercial enhancers:

Gene 32 protein, Perfect Match, Taq Extender, AccuPrime, E. Coli ss DNA binding

unexpected reactioncomponents

DNA dyeslab management

hardware:PCR platform & cups

cycle conditions

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Q 1: Which is an appropriate quantification system?

Q 2: Impact on mRNA and/or microRNA “integrity” on real-time RT-PCR performance ?

Q 3: Impact of extraction procedure on overall RNA integrity ?

Q 4: Impact on quantitative result ?

microRNA

mRNA

Comparison of two platformsDetermination of RNA-Quality and RNA-Quantity

on the basis of their internal algorithms

RINRNA Integrity Number

RQIRNA Quality Indicator

3

RNA quality control via. capillary electrophoresisE-Gram & Electopherogram

Various total-RNA qualities analysed in the Bioanalyzer 2100

Intact RNARIN: 9.5

RIN: 5.6 degraded RNARIN: 2.8

ladder

Fleige & Pfaffl, Mol Aspects Med 2006; Fleige, et al., Biotechnology Letters 2006

marker

marker

marker

marker

5S 18S 28S rRNA

4

Pfaffl, Fleige, Riedmaier; BBE 2008

Concentration input ng/µl51225612864321684210,50,25 51225612864321684210,50,25 51225612864321684210,50,25 51225612864321684210,50,25

Con

cent

ratio

n m

easu

red

ng/µ

l

0,1

1

10

100

BioanalyzerRegression BioanalyzerExperionRegression ExperionConcentration inputRegression Concentration input

Linearity and sensitivity of quantificationtotal RNA input vs. RNA concentration measured

Concentration input ng/µl512 256 128 64 32 16 8 4 2 1 0,5 0,25

Rat

io

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8BioanalyzerExperion

Linearity of 28S/18S rRNA ratio

Pfaffl et al., BBE 2008

5

scattering of concentration measurement (Bioanalyzer)

tissues

kidney muscle WBC heart gut liver

conc

entra

tion

[ng/

µl]

50

100

150

200

250scattering of concentration measurement (Experion)

tissues


conc

entra

tion

[ng/

µl]

50

100

150

200

250

scattering of concentration measurement (Nanodrop)

tissues


conc

entra

tion

[ng/

µl]

50

100

150

200

250

Reproducibility of RNA quantification [100 ng/µl]

- Bioanalyzer 2100

- Experion

- NanoDrop 1000

RNA integrity – 11 degradation steps in heart and intestine tissue

y = 1.07x - 0.08r2 = 0.76

y = 0.87x + 0.81r2 = 0.67

1

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7 8 9 10

RIN

RQ

I

intestineheartidealLinear (heart)Linear (intestine)Linear (ideal)

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Influence of tissue (n=6), extraction (n=6), and chip setup (n=4) on RIN and RQI value

0.960<0.0010.052<0.001liver

0.900<0.0010.379<0.001intestine

0.530<0.0010.135<0.001heart

0.294<0.0010.163<0.001blood

0.170<0.0010.877<0.001muscle

0.925<0.0010.304<0.001kidney

chip runextractionchip runextraction

RQIRIN

p-value

Q 1: Impact on mRNA integrity on real-time RT-PCR performance ?

Q 2: Impact on microRNA integrity on real-time RT-PCR performance ?

Q 3: Impact on quantitative result ?

7

Degradation scale

total RNA extracted from bovine tissues

total RNA extracted from bovine tissues

analyzed in Bioanalyzer analyzed in Bioanalyzer

degradation scale ofmixtures between

good and bad samples

degradation scale ofmixtures between

good and bad samples

analyzed in Bioanalyzer analyzed in Bioanalyzer

artificial degradation

18 S

28 S

28 S

18 S

Marker

Marker

WBC RIN: 9.5

WBCRIN: 2.8

Fleige, et al., Biotechnolgy Letters 2006

RIN 10.0 9.2 9.2 8.6 8.1 7.8 7.2 6.7 6.0 5.0 4.4 4.0

Degradation of extracted total-RNA

18 S

28 S

The intensity of bands decreases with increasing total-RNA degradation

5 S

8

RNA integrity number [RIN]0 2 4 6 8 10

cros

sing

poi

nt [C

P]

5

10

15

20

25

18 S28 Sß-ActinIL-1ß

Impact of RNA degradation on Cq value => mRNA quantification

RNA integrity number [RIN]0 2 4 6 8 10

effic

ienc

y

1.0

1.2

1.4

1.6

1.8

2.0

18 S28 Sß-ActinIL-1ß

Impact of RNA degradation on qPCR efficiencyCorbett Life Science RG-6000

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Effect of degradation in muscle RNAin long RNA (> 200 nt) and small RNA (< 200 nt) fractions

11 artificial UV degradation steps“one-tube” extraction procedure (Qiagen)

analysis of muscle long RNA11 degradation steps on “NANO eukaryote RNA chip”

analysis of muscle small RNA11 degradation steps on “Small RNA chip”

RIN

1 2 3 4 5 6 7 8 9 10

smal

l RN

A c

once

ntra

tion

[pg/

µl]

10000

15000

20000

25000

30000

35000

40000

WBCmuscleliver

y=13369.081+538.98xr2=0.063

y=15711.618+2674.671xr2=0.910p<0.001 y=21379.442+558.875x

r2=0.114

Influence on RIN value on small RNA quantity [pg/µl]

10

RIN

0 1 2 3 4 5 6 7 8 9 10

miR

NA

con

cent

ratio

n [p

g/µl

]

0

500

1000

1500

2000

2500

3000

3500

WBCmuscleliver

y=2680.442‐180.483xr2=0.413

y=3625.62‐201.113xr2=0.339

y=3771.043‐305.179xr2=0.765

Influence on RIN value on microRNA quantity [pg/µl]

RIN

1 2 3 4 5 6 7 8 9 10

miR

NA

/sm

allR

NA

ratio

[%]

123456789

1011121314151617

WBCmuscleliver

y=16.444‐1.412xr2=0.720

y=16.364‐1.400r2=0.791

y=17.851‐1.339xr2=0.669

Influence on RIN value on microRNA quantity [as % of small RNA]

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average slope = - 0.81

n = 6 p < 0.001

average slope = - 1.58

n = 9 p < 0.001

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Which is the best blood sampling methodfor quantitative mRNA & microRNA analysis?

Blood Sampling Which method is the best and most consistent?

Methods for mRNA & microRNA extraction:

5 animals

3 sampling

S1 S2 S3

n =15

ResultsRNA Integrity & RNA yields

NA1.57±0.048.68 ± 0.737.04±0.532.10±0.013.10±0.239LL

6.33±1.741.18±0.101.51± 0.188.87±0.142.01±0.021.68±0.1442.5PAX

NA------4.96±0.581.71±0.0114.32±1.100.35WB

29.13±5.481.23±0.030.34±0.427.37±0.901.95±0.014.53±0.589PI

6.13 ± 0.27------9.45±0.042.02±0.014.44±0.309LY

miRNA %260/280µg/ml bloodRIN260/280µg/ml blood

RNA (< 200 nt)RNA (> 200 nt)total volume

[ml]

extraction method

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WB

RNA Integrity Number [RIN]

0 2 4 6 8 10

Cro

ssin

g P

oint

[CP

]

18

20

22

24

26

28

30

32

34RIN vs 18s RIN vs HIS RIN vs UBQ RIN vs MCSF

ResultsqRT-PCR results and data analysis for WB and PAX method

PAX

RNA Integrity Number [RNA]

6 7 8 9 10

Cro

ssin

g P

oint

[CP

]

14

16

18

20

22

24

26

28

30

32ß-ActinIL-1ßCD14 MCSF 18s

30.29±0.870*31.04±0.552**27.47±0.79425.43±0.96**28.13±0.61COX2

26.78±0.486***25.19±0.21426.09±0.075***25.61±0.11*24.77±0.11NFκB

29.3±0.317***28.17±0.14926.94±0.241***25.53±0.19***28.26±0.14MCSF

27.44±0.305***25.99±0.11726.34±0.198*24.99±0.14***25.9±0,13C1q

28.63±0.501***27.29±0.179*28.59±0.186***27.80±0.23***26.36±0.22C3

27.75±0.406***26.34±0.147*27.11±0.146***25.37±0.1325.74±0.20CD14

25.96±0.704***23.15±0.11125.42±0.123***27.67±0.41***22.34±0.09IL-1β

33.13±0.625***28.92±0.47330.79±0.36027.98±0.53**30.00±0.21Histone

24.97±0.507***22.52±0.12624.84±0.243***23.29±0.22**22.26±0.20UBQ

22.33±0.807***18.85±0.11020.59±0.184**19.28±0.2018.98±0.10β-Actin

22.24±0.956***18.15±0.18021.22±0.350***20.37±0.51***17.28±0.1618S rRNA

LLPAXWBPILY

Mean mRNA expression levels [Cq±sem]

Numbers in bold are equivalent to the lowest mean Cq ± sem values considering each gene and the different extractions;Numbers in italics are equivalent to the highest mean Cq ± sem values considering each gene and the different extraction;Significance comparing all methods in relation to the best method * for P<0.5 ** for P< 0.01 and *** for P<0.001 (n=15)

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36.85±0.56***30.10±0.63*28.85±0.2630.72±0.66***28.41±0.22miR 145

27.57±0.62*26.28±0.9123.91±0.33***24.33±0.67***26.39±0.16miR 101

29.67±0.75***26.01±0.80***22.88±0.3225.55±0.67***22.56±0.18miR 27b

28.50±0.72***26.19±0.56***21.41±0.2325.02±1.24*22.59±0.11miR 181

23.24±0.5626.15±0.64**25.77±0.30**25.96±0.61**23.94±0.13miR 142

29.67±0.61***21.48±0.40***18.19±0.1521.16±1.21**18.07±0.15miR let7 a

24.81±0.85***18.11±0.5915.59±0.24*19.98±1.2618.14±0.15miR16

LLPAXWBPILY

Mean miRNA expression levels [Cq±sem]

Numbers in bold are equivalent to the lowest mean Cq ± sem values considering each gene and the different extractions;Numbers in italics are equivalent to the highest mean Cq ± sem values considering each gene and the different extraction;Significance comparing all methods in relation to the best method * for P<0.5 ** for P< 0.01 and *** for P<0.001 (n=15)

0.7240.32970.467140.9333 / 2Replicates

0.5830.733156.744626.9785 / 4Animal

<0.00112.2552622.2005244.4005 / 4Extraction method

[%] miRNA

0.3091.2653975.4757950.9503 / 2Replicates

0.0017.38023185.64692742.5855 / 4Animal


miRNA Yield

0.8770.1320.0001970.0003953 / 2Replicates

0.0532.6160.003920.01575 / 4Animal


RNA Purity (A260/280 )

0.2651.3864.0558.1103 / 2Replicates

0.1042.1006.14124.5635 / 4Animal


RNA Integrity Number (RIN)

0.8290.1890.9291.8573 / 2Replicates

0.0133.77118.55174.2035 / 4Animal


RNA Yield

PFMSSSn / dfSource of Variation

ANOVA statistics

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(PCA) has been conducted with all microRNA target genes and showed differences between the different extraction groups. LY (red dots) show the best results followed by WB (dark blue dots), PAX (pink dots), PI (green dots) and LL (light blue dots) which shows the most spread cluster.

PCA = Principle component analysisfor small RNA < 200 nt

PCA = Principle component analysisfor messenger RNA > 200 nt

PCA has been conducted with all mRNA target genes and showed differences between the different extraction groups. LY (red dots) show the best results followed by PAX (pink dots), PI (green dots), WB (dark blue dots) and LL (light blue dots) which shows the most spread cluster.

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Summary & Conclusion• Capillary electrophoresis is a perfect tool to measure RNA integrity:

• Bioanalyzer 2100 => RIN algorithm

• Experion => RQI algorithm• 18S/28S ratio is not appropriate for RNA quality control

• For RNA quantification Nano-Drop is recommended !

• qRT-PCR performance is dependent on total-RNA quantity & quality !• RNA quality (RIN / RQI value) is highly tissue dependent !

• good RIN [8-10] for cell cultures and WBC

• lower RIN [5-8] for solid tissues, requiring more homogenization during extraction

• RIN / RQI higher than 5 is recommended (Fleige & Pfaffl, MAM 2006)

• mRNA & microRNA integrity in an ONE tube extraction method:

• mRNA & microRNA quality is extraction method dependent

• mRNA & microRNA quality is highly reproducible within extraction replicates

• Effects of RNA integrity on qRT-PCR results in mRNA and microRNA quantification:• high significant influence on mRNA quantification (higher slope!)

• high significant influence on microRNA quantification (lower slope!)

• minor influence on amplification efficiency

• relative quantification using an internal reference gene, performing the ∆CP approach, can partly circumvent the RNA integrity problematic for mRNA (Fleige & Pfaffl, MAM 2006)

• Appropriate controls or database for microRNA reference genes are missing!

Thank you team !

Thank you for your attention !

mrna & microrna integrity - the key to success · mrna & microrna integrity - the key to...

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