New tools for genetic research:

whole genome microarraysand

customized low density solutions

Dr. Th. RygusApplied Biosystems

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

3

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

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

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

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.

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