whole genome microarrays and customized low density solutions · >rt–vit kti >...
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New tools for genetic research:
whole genome microarraysand
customized low density solutions
Dr. Th. RygusApplied Biosystems
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Integrated Gene Expression Workflow
Biological Validationor Candidate Gene Analysis
Final Target Genesfor Routine Testing
Whole GenomeSurvey or Candidate Gene Discovery
Data Interpretation
Data interpretation
31‘097
50-500
1-100
RN
AEx
trac
tion
& R
Tre
actio
n
# genes
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Gene Expression WorkflowWhole Genome Survey - NEW
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Applied Biosystems Expression Array System
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Components of the SystemOverview
• An Analyzer (model 1700) that can image arrays in:
> Chemiluminescence to: • Measure Gene Expression at very low levels
> Fluorescence to: • Locate, auto-grid and Quality Control of every feature• Normalize every feature, independent of gene
expression signal
• Reagent Kits for labeling, hybridization and detectionof targets:
> RT Kit > RT–IVT Kit> Hybridization/Chemiluminescent Kit
• Human Genome Survey Microarray> designed using Public and Celera data
for all known genes (31,097)
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Hybridization and Detection Devices
• Microarrays held in a disposable chamber for hybridization
• Chamber removed for washing (batch processing)
• ~ 1ml hybridization volume (“swimming pool”)
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Chemiluminescence on theArray
Highest specifity and sensitivity– gene-specific 60mer– Chemiluminescence for low expressed
genes you couldn‘t see before– Two CL read-times (5 and 25 seconds)
for increasing dynamic range
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Overview of the process for the RT labeling Kit
AAAAAAA 3’ mRNA
5’
TTTTTTT 5’ purified DIG labeled cDNA
Reverse transcriptionRNA degradationDNA purification
3’
DIG
DIGDIG
= DigoxigeninDIG
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Overview of the process for the RT /IVT labeling Kit
AAAAAAA 3’ mRNA
5’
(T7 Promoter) 5’ cDNA
3’
Reverse transcription
AAAAAAA - 3’ cRNA
5’(T7 Promoter) 5’ cDNA
3’
AAAAAAA cDNA
5’
2nd strand DNA synthesisRNA degradationcDNA purification
cRNA synthesiscRNA purification
3’
DIGDIG
DIGUUUUUUU 5’ purified DIG labeled cRNA
3’
DIGDIG
DIG
3’
DIGDIG
DIG
3’
DIGDIG
DIG
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DIG Ab
AP
DIG Ab
AP
ChemiluminescentSubstrate
EnhancerEnhancer
Light
Applied Biosystems 1700 MicroarrayAssay
Chemiluminescence Detection:
EnhancerEnhancer
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Fluorescence on the Array
• There is not only Chemiluminescence,but also Fluorescence Detection!
– Two oligos co-spotted(gene-specific 60mer and ICP 24mer)
– Read CL (from 60mer) and FL (from24mer) in same spot
– Normalize, Locate and Quality Control the CL signal by FL signal
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Internal Control Probe (ICP) Concept
Immobilized ICP Oligo(co-spotted in every location)
LIZ® Dye Labeled ICT oligo
LIZ ®DIG
+
Hybridization
DIG
LIZ ®
DIG
LIZ ®
DIG
LIZ ®
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FL image with illumination
Internal Control Probe concept: Read FL and CL in same feature
CL image without illumination
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1,454 Microarray Controls
• Chemiluminescent Controls are used to demonstrate that the CL reaction chemistry is performing well during the assay (DIG labeled oligo co-spotted)
• Hybridization Kit Controls are used to monitor mixing, stringency, and washing during the array hybridization protocol (pre labeled DIG target)
• Labeling Kit Controls are used to monitor enzyme activity and DIG incorporation during the labeling kit protocols– RT (bacterial genes)– IVT (linearized plasmids)
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Celera curatedOnly: 12,559(38%)
Public only: 390 (1%)
AppliedBiosystemswet validation: 1,616 (5%)
Public mapping to Celera:18,638 (56%)
Final Product:31,097 genes33,202 probes
Where do the genes come from?
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52 tissues
Statistical analysisof consistent
probe behavior
Pick BEST PROBEfor each gene
Pick BEST PROBEfor each gene
Labeling Microarrays
Experimental validationof all probes
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All 4 probes are goodChoose strongest signal
Probe Profile across 52-tissues of HCT11041
0
20000
40000
60000
80000
100000
120000
140000
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51
Tissues
CL-
INor
m
HCT11041_1 HCT11041_2 HCT11041_3 HCT11041_4
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Eliminate bad probesChoose strongest signal
Probe Profile across 52-Tissues of HCT10045
-20000
0
20000
40000
60000
80000
100000
120000
140000
160000
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51
Tissue
CL-
INor
m
HCT10045_1 HCT10045_2 HCT10045_3 HCT10045_4
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We can tell exactly what this is.And we have an Assay for it.
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0.1
1
10
100
1000
10000
30 pM 10 pM 3 pM 1 pM .3 pM .1 pM .03 pM 0.01 pM 0.0 pM
[Spike In]
Nor
mal
ized
CL
Inte
nsity
ATP7A-1ATP7A-2ATP7A-3EIF1A-1EIF1A-2EIF1A-3
Absent in Liver
Low Abundance in Liver
Fluorescence25mer
Chemiluminescence60mer
System Performance –Low Gene Expression Levels
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Development Goal: High SensitivityVery Low Sample Size Still Gives Good Results on Microarray
0.1
µg T
otal
RN
A
1.0 µg Total RNA
• Both 0.1 µg and 1µg input into the RT-IVT labeling kit yields products that correlate well.
• Data generated with Stratagene UHR sample.
•Shows data from more than 16,000 genes.
R2=0.94
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Comparison of AB 1700 System with Commercially Available Microarray System
• Same Sample Processed in an Expert Microarray Lab and in Applied BiosystemsFoster City
• Compared Stratagene UHR sample through recommended labeling, hybridization, and detection protocols for both systems
• Compare data from 16,661 genes mapped in common between platforms
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Microarray Comparison
Commercial Array Data
Low Level Expression
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Integrated Gene Expression Workflow
Whole GenomeSurvey or Candidate Gene Discovery
Data Interpretation
Data Interpretation
31‘097
50-500
1-100
RN
AEx
trac
tion
& R
Tre
actio
n
# genes
Biological Validationor Candidate Gene Analysis
Final Target Genesfor Routine Testing
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Real-Time PCR in the 3rd Generation
1995
2000
2004
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Applied Biosystems 7300Real-Time PCR System
• Small footprint• Easy plate loading and
removal („DVD-Player“)
• Cooled CCD– Increased Sensitivity
• Universal PCR conditions• Multicolor Detection
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Currently calibrated Dyes:
SYBR®
FAM™VIC®
JOE™NED™ TAMRA™ROX™
Self-calibrating CCD: use any dye combination ofyour choice on the plate
Adding new dyes without hardware change
1 Excitation Filter 4 Emission Filters
Multicolor Detection
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Large Dynamic Range
9 orders of magnitude demonstrated
FAM
1 Copyof DNA!
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Applied Biosystems 7500Real-Time PCR System
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High-SpeedReal-Time PCR
• 40 cycles in < 40 minutes!• Supports 5–30µL reaction volumes• Convenience of 96well container
– New Low Headspace 96-well ReactionPlates
– New High-Speed Master Mix• “Slow” and “fast” mode
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SYBR®
FAM™VIC®
JOE™NED™ TAMRA™ROX™Cy3®
Texas Red ®
Cy5®
Self-calibrating CCD: use any dye combination ofyour choice on the plate
Adding new dyes without hardware change
5 Excitation Filters 5 Emission Filters
Multicolor Detection
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4 color Multiplex -5 color Detection
FAM™, VIC®, NED™, Cy5® & ROX™ Dye
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Sequence Detection SoftwareData Analysis & Viewing – Relative Quantitation
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ABI PRISM® 7900HTSequence Detection System
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Five Instruments in One
Low Density Arrays – larger number of targets
Low Running Costs– 384-well block
High Through-put– large number of samples
Classic real-time PCR– 96-well block
High Speed (soon)
– Fast PCR block
High Speed
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Versatility and Flexibility by Changeable Blocks
ABI PRISM® 7900HTSequence Detection System
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TaqMan Low Density Array
The Perfect Fusion of Real-Time PCR and Microarrays
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Typical Low Density Array Experiment10-400 Genes
hands-on: 15 min.total time: 3 hours
quantitativestandardized
not more expensive
labeling
Fluorescence Reader
Wash steps
hands-on: 5 hourstotal time: 2 days
not very quantitativenot standardised
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39 384 spots
1 to 8 Samples1 to 8 Samples
12 to 380 Targets(per one card)
12 to 380 Targets(per one card)
1, 2 or 4 Replicates1, 2 or 4 Replicates
TaqMan Low Density Arraywith Real-Time Performance
Assays-on-Demand™ products pre-loaded
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www.allgenes.com
number of Taqman Assayson the shelf ...
Click here tobrowse!
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1 sample
4 samples
2 samples
8 samples
Custom Low Density ArraysConfiguration Matrix12 genes 24 genes 48 genes 96 genes 380 genes
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Pipet cDNA (1-100ng) and Universal Mastermix into sample reservoir of Micro Fluidic Card
TaqMan Low Density ArrayLoading
POP-6TaqMan®
2x PCRMaster
Mix
Applied Biosys
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Heraeus Multifuge 3S Centrifuge Custom Heraeus Centrifuge Buckets(holds up to 3 Micro Fluidic Cards)
TaqMan Low Density ArrayCentrifuge for 2 minutes
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TaqMan Low Density ArrayEight Micro Fluidic Channels
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Following the centrifugation step the interconnecting channels are closed using a Sealing Device
seals the MFC
allows extended handling before thermal cycling
Sealed Card
Sealing Device
TaqMan Low Density ArrayCard Sealing
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Micro Fluidic Cards are now ready to be loaded into the 7900HT system
TaqMan Low Density ArrayRemove Loading Ports
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TaqMan Low Density ArrayBenefits
• Minimal pipetting steps • Streamline reaction set-up• Avoid expense of liquid handling robotics
• Lowest sample input (1ng per channel)• Highest accuracy even on 384-well plates• Dramatic reduction of reagent
consumption• Standardized format
• Direct comparison of results between sites or labs
• Fixed Menu Cards and Custom Cards • Pathway Research, Disease-specific, Process-related
panels etc.
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First customer experiences with the TaqMan Low Density Array
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Results #1: MFC reproducibility
• One sample, split into two aliquots• Configuration: 380 genes
• Aliquots run on two separate days
Data kindly provided by Prof. Jacques HAIECH and Dr Claude PHILIPPEIFR 85, "Biomolecules and Therapeutic innovations„, School of Pharmacy, F-67401 ILLKIRCH, France
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18S rRNA
Results #1: MFC reproducibility
6 target genes
Data kindly provided by Prof. Jacques HAIECH and Dr Claude PHILIPPEIFR 85, "Biomolecules and Therapeutic innovations„, School of Pharmacy, F-67401 ILLKIRCH, France
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Results #2: Routine study
• Study: – 16 samples– 96 genes
• Selected MFC configuration: 2 samples perMicro Fluidic Card
8 cards to run the 16 samples
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Results #2: Routine study
• Study run and finished in only two days– Run template delivered with MFC– RT and eight cards run in two days
(<15 min hands-on time per MFC)– 3072 data points– Final data analysis
done in one minute(single study file)
Customer cDNAs on customized Micro Fluidic Card
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Results #3: fixed menue card
• Demo run: – 2 samples
(T-cells, stimulated and unstimulated)– 96 genes
• Immun profiling card: • First “fixed menu”
Card96 genes known to have implications in immune responseduplicates
IL1A IL1b IL2 IL3 IL4 IL5 IL6 IL7 IL8 IL9 IL10 IL12A IL1A IL1b IL2 IL3 IL4 IL5 IL6 IL7 IL8 IL9 IL10 IL12A IL12B IL13 IL15 IL17 IL18 SCYA3 SCYA19 SCYA2 SCYA5 CCR2 CCR4 CCR5IL12B IL13 IL15 IL17 IL18 SCYA3 SCYA19 SCYA2 SCYA5 CCR2 CCR4 CCR5CCR7 GPR9 SCYB10 SCYB11 CSF1 CSF2 CSF3 STAT3 NFKB2IkB2KB CD3E CD4 CCR7 GPR9 SCYB10 SCYB11 CSF1 CSF2 CSF3 STAT3 NFKB2IkB2KB CD3E CD4 CD8A CD19 IL2RA CD28 CD38 TNFRSF5 PTPRC CD68 CD80 CD86 CTLA4CD154 CD8A CD19 IL2RA CD28 CD38 TNFRSF5 PTPRC CD68 CD80 CD86 CTLA4CD154
HLA-DRA HLA-DRB1 TBX21 TNFRSF18 ICOS NOS2A BCL2 BCL2L1 BAX CD54 SELP SELEHLA-DRA HLA-DRB1 TBX21 TNFRSF18 ICOS NOS2A BCL2 BCL2L1 BAX CD54 SELP SELEHMOX1 PTGS2 REN LRP2 MYH6 RPL3L CYP1A2 CYP7A1 IFNG PRF1 GZMB GNLY HMOX1 PTGS2 REN LRP2 MYH6 RPL3L CYP1A2 CYP7A1 IFNG PRF1 GZMB GNLY
TNFRSF6 TNFSF6 TGFB1 MADH3 MADH7 SKI FN1 COL4A5 C3 TNF LTA ACETNFRSF6 TNFSF6 TGFB1 MADH3 MADH7 SKI FN1 COL4A5 C3 TNF LTA ACE
VEGF CD34 AGTR1 AGTR2 EDN1 ECE1 18S GAPDH ACTB TFRC GUSB PGK1 VEGF CD34 AGTR1 AGTR2 EDN1 ECE1 18S GAPDH ACTB TFRC GUSB PGK1
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Results #3: fixed menue card
18S
HLA-DRA
Beta-Actine
IL-8
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Gene Expression ProfilingComplementary Technologies: High- and Low-Density Arrays
1700 DNA-MicroArrays
7900 MicroFluidic Card
Application Whole-Genome Survey, Gene-centric
Candidate Gene Analysis, Splice Variant Detection
Genes analysed
31.097 12 – 380
Technology Hybridisation Real-Time PCR
RNA-Input 100 ng – 40 µg
1 - 100 ng
Dynamic Range 3-4 logs quantitative
5-8 logs highly quantitative
Flexibility Fixed Targets Different Configurations
Handling, Assay Time
2 days, many steps involved
2 h, just few pipetting steps
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Real-Time PCR?
An Instrument
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Real-time PCR?
ABI PRISM®
7300ABI PRISM®
7500
UniversalConditions
Optimizedand robustReagents
Guidelines for Multiplexing
Guidelines for Quantitation
Primer Express®
Software
TaqMan®
Assays-on-DemandTM
ApplicationSupport
ABI PRISM®
7900HT
ApplicationSolution
TaqMan®
Assays-by-DesignSM