affymetrix low to mid-plex solutions for quantifying genes...

Affymetrix Low to Mid-PlexSolutions for Quantifying Genes & Proteins

2Affymetrix Confidential

Presentation Index

Prepared Presentations

• Corporate Presentation

• QuantiGene 2.0 & QuantiGene Plex 2.0

• QuantiGene ViewRNA

• Solutions for RNAi Gene Silencing and Compound Screening

• QuantiGene for Biomarker Analysis – FFPE & Blood

• QuantiGene for CNV


• QuantiGene 2.0 Reagent System• QuantiGene Plex 2.0 Reagent System

• ViewRNA In Situ Analysis- Single copy mRNA detection in cells- Multiplex detection of mRNAs- Microscope slides & multi-well plates

• QuantiGene Plex 2.0 Reagent System• Procarta Cytokine Plex Kits• Procarta TF Plex Assays• Procarta SH2 Plex Assays

• Stable Cell ines• DeliverX for siRNA• DeliverX for Peptides• Cancer Cell Isolation Kit• Luciferase TF Vectors• Mammalian TF Expression Vectors

• Procarta TF Plex• PD Arrays• Nuclear Extraction Kits• EMSA Kits• Procarta Cytokine Plex Assays• TF ELISA Kits• Cancer Antigen ELISA Kits

Panomics Product Overview

www.panomics.com


Panomics® Technology & Product Foundation

• bDNA: Single and multiplex gene expression assays; ~ From Diagnostics to Discovery and Back Again

bDNA technology invented by Chiron, acquired by Bayer and now Siemens Basis for Siemens FDA-approved Versant™ HIV & HCV viral load diagnostic tests Over 300 publications FDA MAQC study of microarrays and confirmatory quantitative assays published

• Mid-plex RNA, DNA & Protein Analysis: Luminex-bead arrays

• Signaling pathway assays: off-the-shelf & custom

RNA validated assays- ~8,000 singleplex , ~1,000 plex panels TFs- >400 cis elements Cytokines- ~ 218 plex assays for mouse, human, rat, guinea pig & NHP Protein Interaction Domains- genome-wide collections of SH2, SH3, WW, PDZ

Quantitative Gene Expression Assays

QuantiGene 2.0®

and QuantiGene® Plex 2.0


QuantiGene® Applications

• Multiplex Gene Pathway analysis and Microarray data validation

• RNAi gene silencing analysis

• Archival FFPE tissues and Blood

• Biomarkers, Splice Variants and Translocations quantification

• Primary & Secondary Compound and Toxicology screening

• in situ multiplex detection of single molecules

• DNA CNV (copy number variation) quantification


QuantiGene® Assay SystemValue & Benefits

Signal vs. Target Amplification- Direct quantification at source of RNA or DNA - No reverse transcription or target amplification- Avoids biases inherent to reverse transcription or amplification- Avoids false positives (contamination) and false negatives (enzyme inhibition)

Simple Workflow- No RNA or DNA extraction – Work directly from cell lysates or tissue homogenates- Elisa-like workflow & amenable to automation

Accurate & Precise – Detect small differences in gene expression levels- Demonstrated accuracy & precision – FDA lead MicroArray Quality Control “MAQC”Nature Biotechnology Sept 2006

- FDA approved molecular diagnostic assays for HIV and HCV virus – sold bySiemens as “Versant”

- Ideal to validate siRNA knockdown & detect small differences – Dharmaconvalidates all siRNAs using QuantiGene

Flexible Assay Formats, Both Single & Multiplex- Measure anywhere from 1 to 36 targets – mix any match any target in a 96 or 384 well format- Wide range of applications, including Gene Expression HTS, in situ “ViewRNA”, FFPE, blood, DNA CNV


QuantiGene® Reagent Systems- 3 Flexible Formats

Cell LysatesTissue homogenates

- - 200 molecules singleplex-1000 molecules multiplex

QuantiGene Single Gene/Well

Capture Plates

Chemiluminescence

QuantiGene Plex 36 Genes/Well

Capture Beads

Fluorescence

QuantiGene ViewRNA In Situ-Single copy mRNA detection in cells

FISH & CISH


QuantiGene Singleplex Requirements

• Luminometer- Panomics Luminometer- Molecular Devices- Lmax- Promega- Turner Biosystems-

Modulus

Assay Kit

Instruments

• QuantiGene 2.0- 1 RNA or DNA assay/well- 96 well format


QuantiGene Multiplex Requirements

• QuantiGene Plex 2.0 Assay Kit- 3-36 RNA or DNA assays/well- 96 well format

Assay Kit

Instruments • Luminex 200 Flow Cytometer- Bio-Rad Labs- Instrument & S/W- Other Authorized Distributors- Hitachi S/W


QuantiGene View RNA Requirements

• High Content Imaging- Thermo-Fisher-Cellomics/Applied Precision- GE-In Cell Analyzer 1000- Molecular Devices- ImageXpress- Perkin Elmer- Opera- Becton Dickenson- Pathway- TTP Lab Tech- Acumen eX3

• Standard Fluorescent Microscope

High Content Imager

Microscope

Assay Kits

Instruments

• QG View RNA in situ 96 & 394 well assay kit - single molecule detection in single cells (FISH)- 1-4 plex RNAs /cell

• QG View RNA in situ slide assay kit- <10 molecule detection in tissue sections (CISH)

- 1 RNA/cell/tissue section


Gene Expression Quantification Directly from the Source

RNA Isolation

Reverse Transcription

PCR

Eliminates Unnecessary Steps

DATA

SAMPLESCells, Tissue, FFPE,Blood etc.

Signal Amplification Assay

QuantiGene

QuantiGene® Assay Workflow From Sample to Data in a Few Simple Steps


QuantiGene® AssaySpecifications

Singleplex Multiplex

Signal amplification platform 1.0 2.0 1.0

MagneticBeads

2.0

Number of Targets per well 1 1 3-30 3-30

Limit of Detection (RNA copy #) 3000 200 ~24,000 1000

Limit of Quantitation (RNA copy #) 6000 500 ~60,000 2000

Linear Dynamic Range > 3.5 logs > 3.5 logs > 3 logs > 3 logs

Intra-assay Precision (CV) <10% <10% <15% <15%

Inter-assay Precision (CV) <15% <15% <20% <20%

Accuracy of Fold-Change +/-20% +/-20% +/-20% +/-20%

Specificity >99.98% >99.98% >99.8% >99.8%


QuantiGene® Single Gene/Well – 96 Well FormatReagent System Components

Component 1

Coated 96 well Capture Plate

UniversalCapture Probe

Capture Well

Component 2

Target Specific Probe Set

QG Probe Set

CE

LE

BP

Component 3

bDNA Signal Amplification Reagents

SubstrateGlow Luminescence

Amplifier label

Alkaline Phosphatase Conjugated OligosBranched DNA

(Amplifier)


Cooperative hybridization

Simple hybridization

May 2006

QuantiGene® 2.0 Assay Diagram


QuantiGene® 2.0 Assay SystemFold Change Comparison of QG2.0 vs. QG1.0 vs. TaqMan®

y = 1.0176x - 0.0187

R2 = 0.9667

-6.0

-4.0

-2.0

0.0

2.0

4.0

6.0

-6.0 -4.0 -2.0 0.0 2.0 4.0 6.0

QG2.0

QG

1.0

-6

-5

-4

-3

-2

-1

0

1

2

3

4

5

-6 -4 -2 0 2 4 6

QG2.0

TA

Q (

MA

QC

)

y = 0.8449x -0.109R2 = 0.965

Excellent correlation between• QG2.0 and QG1.0• QG2.0 and TaqMan®


QuantiGene® 2.0 Assay mRNAs Quantified by QG and not by TaqMan®

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

C11orf9 GULP1 RECQL4

10 ng/well

RLU

bgk

Brain

UHRR

2.01.7

3.54.1

3.0

5.3

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

GP1BA NR1I3

500 ng/well

RLU

bgk

BRAIN

UHRR

QG 2.0 was able to quantify 5 low abundance mRNAs in two reference samples, Brain RNA and Universal Human Reference RNA, from the MAQC study. Expression of these genes were undetectable (Ct >35) by PCR.


September 2006

MAQC StudyResults Validate the Performance of QuantiGene® Assay


• QuantiGene® outperformed TaqMan® in terms of assay precision and accuracy.

• “The data for the QG were notable because these values encompass system-wide accuracy.” QG performed replicates directly on RNA, TaqMan® only on RT reactions.

• Data are from Canales et al. 2006. Nature Biotechnology 24(9): 1115-1122.

ASSAY ACCURACYPercent difference between actual & predicted values for 118 common targets in samples C and D:

C = 75% A + 25% BD = 25% A + 75% B

ASSAY PRECISION%CVs of measurements made on 224 RNAsin two reference RNA samples:

A = Universal Human Reference RNA (Stratagene)B = Human Brain Total RNA (Ambion)

TAQQGN

TAQQGN

September 2006

MAQC Study of Assay Precision & AccuracyComparison of QuantiGene® & TaqMan®


Panomics® Assay MenuGene expression QuantiGene® Plex RNA KitsCytokines Procarta™ Cytokine KitsTranscription Factor Activity Procarta™ Transcription Factor Kits Phosphotyrosine Profiling Procarta™ SH2 Domain Kits

QuantiGene® Plex and Procarta™

An Exclusive Menu of Luminex® -Based Assays

Luminex:•Multiple Targets •Multiple Samples

ELISA- Multiple Samples

Array – Multiple Targets

Luminex

Multiple TargetsMultiple Samples

• Over 5000 installed units worldwide

• Panomics® provides the widest range of protein & gene assays for the Luminex® System


QuantiGene® Plex Assay A Quick Primer of Luminex® xMAP Technology

5.6μm & 6.5μmMicrospheresPolystyrene & Magnetic

Red & Infaredfluorophores

X MAP Detection System

Analyte coupled to mircosphere2-Laser Flow Cytometer

Red laser - bead classifierGreen laser - reporter quantifier

The Luminex platform is a robust flow cytometer based instrument, installed in > 5000 laboratories.


QuantiGene® Plex Assay Reagent System Components

Bead 1 Bead 2 Bead n

Component 1 - Luminex beads: each bead has a unique spectral signature

Component 2 - QGP 2.0 probe set: A probe set consists of 3 elements.

Capture Extender Probes Label Extender ProbesBlocking Probes

Component 3 – Branched DNA Amplifier

Pre-Amplifier AmplifierBiotin Labeled probe

Streptavidin-PE

Reporter molecule

NOTE: In addition the assay kit contains buffers, reagents, and plates necessary to run the assay.


QuantiGene® Plex 2.0 Assay

Bead 1 Bead 2 Bead n

May 2006

>6,000 Panels


RelRLU

RelMFI

QuantiGene® PlexConcordance with QuantiGene® Singleplex

QuantiGene® Plex

QuantiGene®IL-1

B

IL-8

GA

PD

CS

F2

VE

GF

IL-2

TN

Fa

IL-6

IL-1

0

IFN

G

UninducedInduced

0.1

1

10

100

1000

10000

100000

IL-1

B

IL-8

GA

PD

CS

F2

VE

GF

IL-2

TN

Fa

IL-6

IL-1

0

IFN

G

UninducedInduced

0.1

1

10

100

1000

10000

100000

Correlation Factor = 0.94

• 10 Cytokine singleplex measurementscompared to Cytokine 10-plexmeasurement in a single well

• Inflammatory response stimulated by PMA/LPS

• Samples: 40K cell lysates of U937treated +/- PMA/LPS


QuantiGene® Plex 2.0 Assay Performance: Sensitivity and Dynamic Range

• Data for 17 targets in a representativeplex panel• Dilution series of IVT RNA• Greater than 3 logs linear dynamic range• Sensitivity ~1000 copies (converted from attomoles)

1.0

10.0

100.0

1000.0

10000.0

100000.0

1000 10000 100000 1000000

RNA copies

MF

I (s

ign

al-b

ckg

)ABCB4

NFKB1

RELA

IFNG

UGT1A8

UGT1A9

ABCB11

IL18

CYP2B6

CSF2

TNFSF6

PPIB

VEGF

ABCC2

IL10

GAPDH

ABCB1

Applications:In Situ RNA-Single Molecule Detection


Un-induced Induced

• “80% of mRNAs are present at fewer than 5 copies/cell and 95% of mRNAs at fewer than 50 copies/cell”

• ViewRNA™ is sensitive enough to reliably detect these rare transcripts – unmet need in biology

• Other in situ RNA assays are at best, sensitive only to ~ 20 copies/cell, can’t be multiplexed, aren’tquantitative and are time consuming and complicated

• Applications include: High throughput screening, RNAi silencing, RNA trafficking, experimental medicine and molecular pathology

QuantiGene® ViewRNA™: in situ RNA AssayDetection of Single RNA Transcripts in Single Cells

Multiplex detection: HCV and 18S


QuantiGene® ViewRNA™

Workflow and Features

Total 5hr

• Fluorescent in situ hybridization or chromogenic (FISH or CISH)• Single copy mRNA detection in single cells• Direct detection of gene expression in native cells• Cultured or blood cells: adherent, suspension, tissues• Microscope slides or micro-well plates• Multiplex mRNA detection • Excellent signal-to-noise ratio• Fast and automatable


QuantiGene® ViewRNA™Key Applications

• RNAi Delivery and Knockdown

• Biomarker Studies

• Reporter Gene Screening – high throughput screening

• Transcriptional Heterogeneity

• Molecular & Experimental Pathology – clinical tissue application

• Complements immunohistochemistry (IHC)

• Stem Cell Differentiation – gene profiling stages of differentiation

• Cell Biology, Neurobiology, etc.


QuantiGene® ViewRNAAssay Validation – 18S rRNA

Simultaneous Signal Amplification and Background Reduction

16X AMP 1X AMP10 m

Z Z

Left set Right set 18S sense probe

Z Z

Above: Nuclei stained with DAPI (Blue) – Cytoplasm ViewRNA 18S rRNA (Green).18S rRNA is only detected in the presence of both LEs left & right (ZZ) panel “a” vs. panels “c & d” or using antisense probe

sets (panel a) vs. sense probe sets (panel e). Note: Using constant camera imaging panel a vs. b = 15.95X increase in signal a>b.


Her2 and IL-8 genes are detected in the chromosomal DNA of HeLa and SKBR3 cells using target probes against Her2 (Green) and IL-8 (Red) DNA, respectively. The nuclei were counter-stained with DAPI (Blue). Each dot represents a single DNA copy. Her2 DNA is amplified in SKBR3 cells.

Resolution down to 1KB

QuantiGene® View Detection of Single DNA copy


QuantiGene® ViewRNADetection of Single Copy mRNA

Assay AV. copy # /cell

ViewRNA™ 5.4

QG® 2.0 6.4 ± 2.3

HeLa SKBR3

HE

R2-

C/1

8S r

RN

A/D

AP

I

HE

R2/

18S

rR

NA

/DA

PI

Top left: Her2 ViewRNA probes to processed mRNA (Green) – 18S rRNA ViewRNA (red) – Nuclei DAPI (Blue).

Bottom left: Her2 ViewRNA probes to intron unprocessed of RNA (neg. control). Note: 18S rRNA positive control (Red).

Top right: Comparison of Her2 expression ViewRNA counting dots (RNA transcripts) vs. QuanitGene using IVT standard curve.

Bottom right: Distribution of dots (RNA transcripts) per cell - 200 cells counted under microscope.


QuantiGene® ViewRNA™ ApplicationTranscriptional Heterogeneity 22

0 hr 0.5 hr 1 hr 2 hr 4 hr 8 hr

IL-8 / 18S / DAPI

IL-6 / 18S / DAPI

• Time course of PMA induction of IL6 & IL8 in HeLa cells• Induction kinetics at the single cell level


QuantiGene® ViewRNAMultiplex mRNA Detection – IL6 & IL8

IL6 (Green) & IL-8 (Red) in PMA induced HeLa Cells – Nuclei DAPI (Blue)

IL-8

IL-6

IL-6 & IL-8

DAPI

Heterogeneous expression of IL6 & IL8 in HeLa cells


QuantiGene® ViewRNAAutomation of HTS 96/384 plates

Treat cells Fix cells* Permealize cells Hybridize cells 40ºC 3 hours *

Amplify & label cells40ºC 1 hour

Signal stable for 48hr

DAPI stain cells*

*Stop assay (optional)

Scan plateImage Analysis

5 Hours from Fixation to Image Analysis QG View is an Alternative Method to Gene Expression Reporter Assays

Validated imaging systems:• CellWorX Fx/API• inCell 1000/GE

• ImageExpress Micron/Molecular Devices• Opera/Perkin Elmer

• Acumen eX3-3 lasers/TTP Lab Tech

Validated software:• CyteSeer 1.04/Vala Sciences

• MetaXpress/Molecular Devices• inCell Investigator/GE

All steps have commercially available automation options – semi or fully automated


QuantiGene® ViewRNAQuantification of IL-8 Expression in PMA Induced HeLa Cells

HeLa cells cultured on the poly-lysine coated glass bottom 384-well plate, treated with PMA for 4hr and processed. The images were acquired using ArrayWoRx MF (Applied Precision) and analyzed using “CyteSeer ViewRNA”Software (Vala Sciences).

10ng PMA/well3.33ng PMA/well

1.11ng PMA/well0.37ng PMA/well0.12ng PMA/well

PMA Induction of IL-8 in HeLa Cells

1

6

11

16

21

26

31

36

41

0.01 0.10 1.00 10.00

PMA (Log of ng/well)

Med

ea

n R

NA

do

t c

ou

nts

/ce

ll

PMA Induction of IL-8 (Green) Transcription in HeLa Cells.Nuclei stained with DAPI (Blue)

Dose Response


QuantiGene® ViewRNAsiRNA Knockdown HPRT- HeLa Cells

HPRT (Green)ACTB (Red)

Nuclei DAPI Stained (Blue)

0nM siRNA 0.04nM siRNA 10nM siRNA


QuantiGene® ViewRNAChemiluminescence – Reporter Gene Assay Format

No Probe HPRT PPIBHER2 PGK1

Label Probe-AP + AP Chemiluminescent

Substrate

QG ViewRNA - AP Label Probe

0

20

40

60

80

100

Neg -

no p

robe

Neg -

18Sen

se

Neg -

HCV

HER2

HPRT

PPIB

PGK1

RL

U

400X amplifier

Well

RNA

Alkaline phoshatase conjugated oligos

SubstrateGlow Luminescence

Above: HeLa cells hybridized with multiple QG probe sets - Chemiluminescence signal from entire well of a 96-well plate

Below: HeLa cells hybridized with multiple QG probe sets – Fluororescence measuring mRNA signals (Green) in individual cells.

Chemiluminescence – LMax Luminometer

Quantify “native” gene expression directly from any adherent cell type – no genetic engineering of cells


Detection of endogenous genes in HeLa cells

• 4000 HeLa cells per well in 384-well plate.

• Probe sets for various genes were used for

detection.

No Probe Her2 PPIB GAPDH

0

50000

100000

150000

200000

250000

300000

no probe Her2 PPIB GAPDH

Target probe

RL

U


QuantiGene® ViewRNAMouse C2C12 Myoblast Cell Differentiation to Myotubes

Bright field images of C2C12 differentiation at T = 0, 2 and 5 days in 96 well plate.

T = 5 days

T = 2 daysT = 0


QuantiGene® ViewRNAIncreased Expression of Myogenin Expression in C2C12 Myotubes

T = 5 days

T = 2 daysT = 0

Myotubes expressing myogenin

Myotubes expressing myogenin

C2C12 cells cultured in a 96 well plate were formaldehyde fixed at 0, 2 and 5 days differentiation.

The fixed cells were permeabilized and ViewRNA probesets were used to detect Myogenin mRNA.

DAPI nuclear stain (Blue) & Myogenin mRNA (Green).


QuantiGene® ViewRNAAffymetrix Gene Chip Analysis of Purified Axons

A. Enriched expression includes translation, mitochondrion intracellular

transport, and cytoskeleton genes. Notably, there are many transcripts

not included in these enriched functional categories.

B. Venn diagram showing overlapping transcripts in the naive matured

cortical axons and adult injury-conditioned DRG axons identified.


QuantiGene® ViewRNA – Validation of Affymetrix GeneChip Data FISH Localization & Quantification of mRNA Transcripts in Axons +/- Injury

ViewRNA shows the localization of transcripts for 3 genes in matured axons, their change following injury, and validates the findings of the Affymetrix GeneChip studies.

A. FISH results showing mRNA puncta (Green) present in cortical axons. The axons are also immunolabeled with tubulin(TUBB) (Red).

B. Number of puncta per area of naive axons, showing a similar mRNA expression level trend as the Affymetrix GeneChipanalysis.

C. Normalized puncta levels changed significantly (*) following injury.

ViewRNAA.

B. C.


Breast Tumor

20XColon Tumor Prostate Tumor

Lung Tumor

QuantiGene® ViewRNAHuman FFPE Archived Tissue Sections - Her2 Expression


QuantiGene® ViewRNAAnalysis of Her2 Expression in Breast Cancer Progression


QuantiGene® ViewRNABreast Cancer – Differential Expression of Her2 mRNA

Her

2 (B

rig

ht

fiel

d)

High Expression Low Expression

Top left: image of breast cancer tissue expressing high level of Her2 in cancer cells.

Top right: image of second breast cancer tissue showing low expression of Her2 in the cells.

Bottom: QG2.0 analysis confirms the high (top left)low expression (top right) of Her2 in the tissues.


Breast cancer FFPE tissue section Her-2 / Gill’s Hematoxylin

20X

QuantiGene® ViewRNAHuman Breast Cancer FFPE Tissue Section – Her2 Expression


Breast cancer FFPE tissue section EGFR / Gill’s Hematoxylin

20X

QuantiGene® ViewRNAHuman Breast Cancer FFPE Tissue Section – EGFR Expression


QuantiGene® ViewRNACancer & Normal Breast FFPE Tissue – Her2 Expression

Tumor Adjacent tissue 1.5cm from tumor

Her2 (ERBB2) mRNA in Breast Cancer FFPE sections was labeled using QuantiGeneViewRNA for FFPE.

Left: image of breast cancer tissue expressing high level of Her2 in the cancer cells (arrows).

Right: image of adjacent breast tissue showing no expression of Her2 in the cells.


QuantiGene® ViewRNANormal & Cancer Breast FFPE Tissue - CDK19 mRNA Staining

CDK19 (Cytokeratin 19) is an epithelial cell specific marker that can be used to identify

epithelial cells in a mixture of cells in tissues.

20X

Bright field CDK19

Hematoxylin stained Fluorescent images

Adjacent Normal Tissue

Breast CancerTissue

Grade III / Stage IV

Top right: Normal tissue - exclusive CDK19 stained epithelium with healthy ductal structures. Bottom right: Cancer tissue – No ductal structures - CDK19 staining of cancerous epithelial cells

infiltrating surrounding tissue.


QuantiGene® ViewRNA - Human Liver FFPE View RNA 30’ Incubation vs. FFPE Radioactive 3 Week Exposure

Comparison of QuantiGene ViewRNA detection sensitivity to radioactive labeled probes.

Left: detection of IGFBP-3 mRNA expression in human liver Kupffer cells with S35 UTP labeled probe. Arrows indicate the positive signal detected after exposing the x-ray film for 3 weeks.

Right: detection of IGFBP-3 mRNA in human liver Kupffer cells using FFPE ViewRNA assay with 30 min chromogenicsubstrate incubation.

Arany E., et al J Clinical Endocrinology & Metabolism FFPE ViewRNA


QuantiGene® ViewRNAMouse Sagittal Brain Section – Drd1a – Dopamine Receptor D1A


QuantiGene® ViewRNAMouse Sagittal Brain Section – Dopamine Receptor D1A


RNAi Gene Silencing and Compound Screening


Panomics® Solutions throughout the Drug Discovery & Development

Exploratory Preclinical Clinical Post Launch

Launch

• Target & microarray validation• RNAi knockdown quantification• GE-HTS primary screening• Secondary compound screening

• ADME & Toxe.g. Transporters, P450s

• Biomarker Validation, Clinical Trials• e.g. Gene expression in FFPE & Blood

• Companion drug tests,Theranostics


Target Identification

Hit Finding Hit Validation Hit to LeadLead

OptimizationDrug

Candidate

Drug Target

Compound collection

HTS

In silicoScreening

Secondary assays

Iterative Chemistry

Safety

Animal Model

Theranostic

Diagnostic Biomarkers

Panomics Solutions throughout Discovery & Development of Disease Tests and New Drugs

Panomics QG ProductsValidation Applications• Microarray validation

• Target validation• RNAi kd quantification• Biomarker Validation

Affymetrix ProductsWG expression profiling:

• 3’ IVT• WT Gene Plate Array

• miRNA

Panomics QG ProductsScreening Applications

• RNAi knockdown quantification• GE-HTS primary screening

• Secondary compound screening

Affy GeneChip Products WG expression profiling

• Screening Array

Panomics QG Products• ADME & Tox

e.g.Transporters,P450s• Cytokines Profiles

Affymetrix ProductsWG expression profiling


• miRNAWG Genotyping

• GWAS- SNP6.0 human• MyGeneChip for animals

• Axiom

Panomics QG Products Biomarker & Clinical Trial

Applications• Gene Expression Signature

• Copy Number Quantify• FFPE & Blood

• Cytokine profiles• Companion drug tests

Affymetrix ProductsWG expression profiling


• miRNAWG Genotyping

• SNP 6.0- association & CN• Cyto chips

• Axiom• DMET chips


•Transfect Cells (IL8 siRNA)•Rx PMA•Lyse Cells

Measure basal and knockdown expression level of IL8. Normalize against Cyclophilin.

HeLa Cell Culture

•Mock Transfect Cells•Rx w/ vehicle•Lyse Cells

Aliquot to Capture Plate* Aliquot to Capture Plate*

HeLa Cell Culture

*Cell number and cell treatment are uniform across all Capture wells..

Experimental design to test the precision of QuantiGene®

in quantifying small differences between ‘knock-downs’

QuantiGene® Assay for RNAi Screening & ValidationExquisite Precision in Quantifying Knock-downs


QuantiGene® Assay for RNAi Screening & ValidationPrecisely Quantify <10% Differences in RNAi knock-downs

89% knockdown 94% knockdown

0

50

100

150

200

250

300

350

RL

U IL-8

Untreated HeLa

PMA induced HeLa

IL-8 siRNA: 4 hrtreatment

IL-8 siRNA: 8 hrtreatment

Cyclophilin

TreatmentUntreated HeLaPMA induced HeLaIL-8 siRNA: 4 hr treatmentIL-8 siRNA: 8 hr treatment

Average STD % CV Average STD % CV0.69 0.03 5% 39.19 2.54 6%

305.90 36.37 12% 48.55 3.47 7%32.95 2.76 8% 40.21 4.30 11%17.27 2.62 15% 49.50 4.20 8%

IL-8 Cyclophilin

Δ 5%

Raw data of 89 & 94% knockdown of IL-8


QuantiGene® Assay for RNAi Screening & ValidationPrecisely Quantify <10% Differences in RNAi knock-downs

Treatment Average STD % CVUntreated HeLa 0.018 0.0005 3%PMA induced HeLa 6.307 0.6884 11%IL-8 siRNA: 4 hr treatment 0.823 0.0705 9%IL-8 siRNA: 8 hr treatment 0.350 0.0535 15%

Ratio (IL-8 / Cyclophilin)

87% knockdown94% knockdown0.000

1.000

2.000

3.000

4.000

5.000

6.000

7.000

Untreated HeLa PMA induced HeLa IL-8 siRNA4 hr post-Rx

Rat

io (

IL-8

/ C

yclo

ph

ilin

)

IL-8 siRNA8 hr post-Rx

350 fold induction

87% knockdown 94% knockdown

Δ 7%

Normalized data of 87 & 94% knockdown of IL-8


QuantiGene® RNAi Applications Dharmacon validation of siRNAs by QG 2.0

Why QuantiGene?

• Precisely quantify knockdown efficiency of individual siRNA’s.

• This precision allows for optimization of the siRNAcomposition & concentration of SMARTpools®members.

• No RNA isolation or purification high precision allows fine for tuning

Figure 4. mRNA knockdown of human Cyclophilin in cultured cells detected using branched DNA. Cyclo 1, 2, 3 and 4 represent the individual duplexes that compromise the SMARTpool, The non-specific control duplexes are identical in length and similar in GC content to the siRNA targets, but have no significant homology to any human or mouse mRNA sequence. The duplexes and SMARTpool are 100 nM final concentration

Technical Bulletin #006 www.dharmacon.com

• Dharmacon validates their siRNAs using QG2.0

• Dharmacon has the siRNAs and Panomics has the validated QG probe sets.


September 2005

QuantiGene® Plex Application: siRNA Knock-downEffects of IL-8 siRNA Knockdown on 11 Pathway Genes


QuantiGene® ViewRNATransfection and Untreated HeLa Cell Controls – HPRT

0nM siRNA -Transfected Cells only – No Transfection




QuantiGene® ViewRNAsiRNA Knockdown HPRT- HeLa Cells



0nM siRNA 0.04nM siRNA 10nM siRNA


Affymetrix Gene Expression WorkflowQuantiGene® siRNA Validation of GeneChip® Data

Figure 2. ASNS expression following synthetic siRNA transfection of ovarian cancer cell lines. A, branched-DNA assay for ASNS mRNA 48 h after transfection of siNeg, siASNS.1, or siASNS.2. ASNS levels for a given sample were normalized to PPIB levels for that sample.

Figure 1. ASNS transcript levels were measured using four different microarray platforms: cDNA array, Affymetrix Hu6800 array, Affymetrix U95 array, and Affymetrix U133 array.

Philip L. Lorenzi et. al., Mol Cancer Ther 2006; 5(11). November 2006

Affymetrix Gene Chips were used to molecular profile NCI-60 cancer cell lines and QuantiGene to validate L-ASP cytotoxicitylinkage to ASNS expression by siRNA knock-down demonstrating the possible use of L-ASP for treatment of subsets of

ovarian cancers with ASNS as a biomarker for patient selection.


QuantiGene Plex® 2.0 ApplicationAmgen Case Study: in vivo Screening

Amgen Conclusions:

• Superior data to qPCR

• QGP reduced # of plates by14-fold

• Allows easy comparison of protein release and gene expression across different tissues, samples, and models which improves efficiency of In vitro compound profiling

• 14-plex QG® Plex vs. qPCR for in vivo quantification of mRNA profiles in LPS-induced mice spleens

• QuantiGene® Plex 2.0 was used to analyze 156 mRNAs in 552 samples from the CIA animal model.

• Both protein release from cell supernatants & mRNA from cell lysates of the same sample quantified


QuantiGene® Plex ApplicationSecondary Screening of Compound Treated Cell Lines

• HUVEC cells seeded at 10,000 cells/well• Treated with IL-1β at various doses for 24hr• QG Plex - cell lysates used directly

Data courtesy of GSK Pharmaceuticals


QuantiGene® Plex Tox Screening Application Drug Induction of Transporters & P450 Metabolizing Enzymes

0.0

1.0

2.0

3.0

4.0

MR

P1

MR

P2

MR

P3

Pgp

MD

R3

BS

EP

BC

RP

OC

T1

OA

T2

OPZ 100uM BNF 50 uMDex 10 uM PB 100 uMRif 10 uM

05

10152025303540455055606570

CY

P1

A2

CY

P1

B1

CY

P2

A6

CY

P2

B6

CY

P2

C9

CY

P2

C1

9

CY

P2

E1

CY

P3

A4

CY

P3

A5

OPZ 100uM BNF 50 uMDex 10 uM PB 100 uMRif 10 uM

P450 gene response to prototypical inducers as expected

ABC Transporter Panel- Drug Induction in Human Hepatocytes

Cytochrome P450 Panel- Drug Induction in Human Hepatocytes

Relative gene induction detectable post drug treatment for both panels


RNA Quantification from Archival FFPE Samples


QuantiGene® FFPE Reagent SystemAssay Workflow and Benefits

BioTechniques April 2006

No RNA purification Works directly from tissue homogenates

No reverse transcription or amplification Avoids biases inherent to RT & amplification

Insensitive to chemical modifications Modification of RNA basesRNA-protein cross-links

Insensitive to RNA degradation Efficient capture of even highly degraded RNA

No Chemical De-waxing required.


QuantiGene® FFPE AssayQuantification is Unaffected by Formalin Fixation

RNA was not degraded, as shown on an agarose gel.

1hr+

1h

r -

16h

r-

16h

r+

Co

nt r

ol

28S18S

17

19

21

23

25

27

29

1h- 1h+ 16h- 16h+ 1h- 1h+ 16h- 16h+ 1h- 1h+ 16h- 16h+

ACTB PPIB GAPD

Ct

0

100

200

300

400

500

1h-

1h+

16h

-

16h

+

1h-

1h+

16h

-

16h

+

1h-

1h+

16h

-

16h

+

ACTB PPIB GAPD

RL

U

qPCR: Up to 15-fold reduction in signals

QuantiGene®: No reduction in signalsFix in formalin

QuantiGene

qPCR

HeLa cells

Fixed cells

Total RNA

cDNA

RT

Isolate RNA


QuantiGene® FFPE AssayQuantification is Less Affected by RNA Degradation

Log-transformed standard curves for a representative target using serial dilutions of intact (0 minute) and NaOH-degraded (9-minute) IVT RNA pools demonstrate

• Linear dynamic range exceeds 3 logs even with degraded RNA

• Only 2-4 fold reduction in signals from highly degraded RNA

0 9

0.5kb

NaOH (minutes)


QuantiGene® FFPE Assay: Quantity-QualityAssessment Assay Applied to 3 & 14 Yr Old Samples

Quantity Assessment:• 18S Ribosomal DNA probe sets are used to quantify the

number of cells in a FFPE sample

Quality Assessment:• 28S Ribosomal RNA probe sets are to test the quality of the

RNA in a given FFPE sample

Ratio 28S rRNA /18S rDNA:• Ratios vary from 0.1-2.0• Depending on ratio more or less sample is needed to quantify

mRNA expression of equal copy number/cell

Multiple Housekeeper Gene Probe Controls:• 3-5 housekeepers combinations enable “geometric mean”

normalization for improved data analysis• >20 housekeeper’s to select from

See next slide for the correlation between the same 3 &14 yr. old samples using 12 Housekeeping

genes using equal amounts of sample input

28S/18S = 1.0

28S/18S = 1.0 28S/18S = 0.25

28S/18S = 0.25


QuantiGene® FFPE Assay Quantification of Housekeeper Genes in 3 & 14 yr FFPE Samples

R²=0.9938

Excellent correlation of RNA signals for 12 housekeeping genes: 3 vs. 14 yr. old FFPE lung tissue samples from different patients

3N 3T 14N 14T

28S

18S

Agarose Gel

See next slide for comparison of same samples using a reported biologically relevant target mRNAs


QuantiGene® FFPE Assay: Quantification of Biologically Relevant mRNAs in 3 & 14 Yr FFPE Samples

0

1

2

3

4

5

LDHA RPL19 RPL32

Fold Change

3 T /3N

14T/14N

RNA from 3 and 14 year old FFPE samples of lung tumor and adjacent normal tissues run on an agarose gel.

In agreement with the literature, QuantiGene

detected 2-fold induction of LDHA RNA In

tumor relative to normal tissue even in highly

degraded 14-yr old sample (Beer et al. 2002).

1

10

100

1000

0.1 1 10 100 1000

Sample Input (ng equivalent)

RL

U

3T LDHA 3T RLP32 14T LDHA 14T RLP32

Raw data for LDHA and RPL32 RNAs from dilutionseries of 3T and 14T FFPE tissue homogenatesshowing linear dose response.

3N 3T 14N 14T

28S

18S


QuantiGene® FFPE Assay System Sample Types and Typical Amounts of Starting Material

•~20K cells• 28S/18S = 1.0•~10-15 RNA copies/cell• 300 assays if QG 2.0

Cell Number RNA Quality RNA Expression

Number of Assays


FFHC Case Study Comparison of QG® & TaqMan®

• Cancer and normal tissues from the same FFPE blocks were macro dissected and dissolved in Homogenizing Solution.

• A panel of 14 prostate cancer genes was measured in cancer and adjacent normal tissues.

• Values for each gene were normalized to a housekeeping gene (RPL19).

• The ratio between cancerous and normal tissues is calculated and shown in a 5-tiered categorical scale.

• “In macro-dissected tissues from 9 – 13 year old blocks with poor RNA quality, the QuantiGene® 2.0 Reagent System correctly identified the over-expression of known cancer genes (arrows).”

• “In conclusion, the QuantiGene® Reagent System appears to be well suited for clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels.”

Knudsen et al J Mol Diagnostics 2008


QuantiGene® Plex FFPE AssayBiomarker Validation - Affymetrix GeneChips to QGP Assays

AffymetrixGeneChip®

PanomicsQuantiGene® Plex


QuantiGene® Plex 2.0 FFPE Assay Her-2 RNA Expression in Breast Cancer FFPE Biopsies

Soonmyung Paik - NEJM March 27, 2008


Reliable Gene Expression Profiling of HumanFFPE Tumor Xenografts

Normalized FFPE Sample Data

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

GLU

D1 (9

)

UBE2D3

(11)

IFI4

4 (1

8)

IFIT

1 (1

9)

CTDSPL (2

2)

IFIT

3 (2

4)

OAS1

(25)

ZNF273 (3

0)

MX1

(34)

IGF1R

(39)

STAT1 (44)

ABCD3 (5

1)

FLNB (53)

ISG

15 (6

1)

Rel

ativ

e F

luo

roes

cen

ce U

nit

s

NU61 DOXO 3 NU61 Control 1 SCC61 C2 2X SCC61 C1 2X

Data courtesy of Dr. Andy Minn, U. of Chicago.

• Tumor xenograft model of head and neck squamouscell carcinoma

• SCC-61, parental cell line, radiosensitive (yellow and green bars).

• Nu-61, derivative cell line, radiation resistant (blue and red bars).

• Circles indicate genes of the IFN-pathway known to be up-regulated in resistant cells.

• Gene expression data were normalized to PPIB.


QuantiGene® 2.0 FFPE Companion Diagnostic Application A Collaborative Study with Lilly & Siemens on Prostate Cancer

Panomics developed the assay, performed validation and Phase I clinical assays in house

J Clin Invest. 2007 Sep;117(9):2638-48. Therapeutic suppression of translation initiation factor eIF4E expression reduces prostate tumor growth without toxicity.

Phase II clinical trials – samples are needle biopsies in FFPE

Panomics is transferring the FFPE assay to Siemens Diagnostics reference lab for Phase II and III clinical trial assays


RNA Quantification

from Blood Samples


PAXgene

QuantiGene® Blood Reagent SystemAssay Workflow and Benefits

July 2006

No RNA purificationWorks directly form whole blood or PAXGene® tubes

No Reverse transcription or amplificationAvoids biases inherent to RT & polymerasesAvoids enzyme inhibitors found in blood

No Globin RNA depletion required

Can Multiplex up to 30 genes/well

Small volume of blood required- typically <15 ul


QuantiGene® Blood Assay SystemInsensitive to Interference from Blood Components

QuantiGene® Blood is Insensitive to Interference from Blood Components. Detection of E.coli dapB gene in vitro transcripts (IVT) in the presence (diamond) and absence (square) of lysates from 20 µl whole blood, using QuantiGene® Blood. Mean + SD values are graphed. R² values for both curves are 0.99

.


QuantiGene® Blood Application Quantify mRNAsFrom Sub Populations of Whole Blood Samples

0

20

40

60

80

100

120

0 5 10 15 20 25 30 35

Microliter Whole Blood

Net R

LU

CD66d (Neutrophil)

CD3E (T cell)

CD61 (Platelet)

Quantitative Detection of Cell Marker Genes in Heparinized Whole Blood Using the QuantiGene Blood Reagent System.

Quantitative detection of mRNA expressed in minority blood cell populations using a single drop of whole blood. Fresh, heparinized whole blood was lysed and assayed for various cell markers using target-specific Probe Sets. The mean CVs were 6%.


Foxp3 expression in 10ul mouse whole blood

-113579

111315

ctr Ig#1

ctr Ig#2

ctr Ig#3

Ig #1 Ig #2 Ig #3 Ig #4 Ig #5

Mouse ID

(Fo

xp3/

GA

PD

H)*

100 Ig stimulated

Prebleed

QuantiGene® Blood Application Analysis of Mouse Whole Blood

• Study Design: An experimental antibody stimulates the proliferation of regulatory T cells.

• Forkhead box P3 (FOXP3) is a marker for regulatory T cells.

• Five mice were treated with the stimulatory antibody (Ig) and three mice were treated with a control antibody (ctr Ig).

• Blood was drawn before stimulation (prebleed) and 6 days after stimulation (Ig stimulated) from the same animal.

• FOXP3 RNA levels were normalized to GAPDH expression.

• Regulatory T cells were induced in 4 of 5 mice treated with stimulatory antibodies.

Data courtesy of Dr. Y Liu


QuantiGene® Blood Assay SystemComparison of Whole Blood vs Pure RNA

• Correlation of gene expression in whole blood and

RBC-lysed blood.

• Lysates of 20ul of whole blood and equivalent

volume of RBC-lysed blood were assayed in QGP

• Gene List: IL2, TNFa, IL10, IL6, IL1B, IFNg, IL8,

CSF2, GAPDH, RELB, A20, CDKN1, NFKB1,

NFKB2, RELA, NFKBIA, BAK, FASL, FAS, RAFTK,

BAD, BCL-2, IL6R, BCL-XL, ACTB and CFLAR

1

10

100

1000

10000

1 10 100 1000 10000

Whole Blood

RB

C-l

yse

d B

loo

d

BCL-XL

R2=0.998

1

10

100

1000

10000

1 10 100 1000 10000

Whole blood lysate, 20ul Whole Blood

Wh

ole

blo

od

RN

A, 1

60u

l blo

od

R2=0.956

Slope=2.3378

Expected slope 8, actual 2.3RNA extraction efficiency: 2.3/8 = 29%

MFI

MFI

MF

I

MF

I

• Excellent correlation betweenwhole blood and RBC-lysed blood.

• Very good correlation betweenwhole blood and purified RNA.

BCL2L1


QuantiGene® Plex Blood Reagent SystemAffymetrix GeneChips to QuantiGene Plex Assays

Excellent Discriminators for Multiple Sclerosis found in whole blood classification Models with 98.41% Accuracy (7-gene) &

100% accuracy (12-gene)

*

*False Discovery Rate Correction

>300 GenesCovered by

PCT Filing

DxModels

7-gene

12-gene

>10k GenesSignificantlyDifferentially Expressed (p ≤ 0.05)

>600 GenesSignificantlyDifferentially Expressed (p ≤ 0.001)

FD

R*

Bio

log

ically Releva

nt

& S

tatis

tically

Prio

ritized

AffymetrixGeneChip®

PanomicsQuantiGene® Plex


QuantiGene® Plex Blood ApplicationMultiplex Quantification of Gene Expression in Blood

Multiplexed RNA measurements in whole blood using the QuantiGene® Plex Blood Reagent System. Fresh heparinized whole blood was incubated at 37°C for 125 minutes with or without 10 µg/mL LPS. Sixteen microliterswere removed and assayed for cytokine gene expressions. Control is blood sample at t=0. * Significantly different from Control (P<0.01). Mean + Std. Dev. are shown.

GAPD IL-1B IL-6 TNFa IFNg IL-10 IL-2 CSF2IL-8

QuantiGene® Applications:

DNA Copy Number VariationQuantification


QuantiGene® Plex DNA Assay Single Copy Gene Resolution Discriminates 0.5 to 6 Copies

0

10

20

30

40

50

60

70

80

90

0 1 2 3 4 5 6 7

Copy Number Per Cell

Sign

al(M

FI)

DapB gene / 10,000 – 140,000 copies of Bacillus genomic DNA are spiked into 20,000 HeLa cell lysates. The data suggests that QGP DNA assay differentiates from 0.5 to 6 DNA copies per cell.

Bacillus DapB Gene


Crude extracts tested in 96-well high throughput format. Cry1F is normalized to Sus1.

This demonstrates the utility in tracking and determining the target gene copy number between various corn plants for uses in target gene tracking,

transgene development, GMO testing, zygosity analysis, and molecular breeding.

QuantiGene® DNA Assay Cry1F transgene – B73 Maize Zygosity HTS

Collaboration with Dow AgroSciences


QuantiGene® 2.0 DNA DNA Copy Number Assay Validation

0

510

1520

25

3035

40

1 2 3 4 5 6 7 8 9

wild-type "late-responders" "early-responders"

Rat number and phenotype

Norm

aliz

ed G

OI R

LU

Detection of Human Transgene SOD1 in Rat Animal Model of ALS

• Transgene copy number determined by QuantiGene® 2.0 assay correlates with observed phenotype.

• The one exception (#5) was confirmed by PCR to be a mis-classified, non-transgenic animal.

• “Your analysis was spot-on! Your technique is a global way of predicting disease onset in transgenic models of neurological disease. The importance of being able to predict disease onset when designing experiments with these animals cannot be overstated. ” [Prof. Clive Svendsen, U. Wisconsin]

Non-transgenic?


QuantiGene® DNA AssayDetection of Human Transgene SOD1 in Rat Animal Model of ALS

Extended Study

The transgenic SOD1G93A (or ‘Howland’) rats develop neurological and neuropathological symptoms reminiscent of human ALS, i.e. progressive loss of motoneurons leading to paralysis and death. At the beginning of the colony, symptoms of the model disease

appear at 95-125 days of age, and the animals survived till 120-145 days of age. Thereafter a change in the disease phenotype & SOD1 DNA copy number occurs, where approximately 1/3 of the rats displayed much slowed disease progression.

Human Sod1/Rat Prolactin

0

5

10

15

20

25

30

35

40

WT

WT

WT

WT

WT

SOD1

SOD1

SOD1

SOD1

SOD1

SOD1

SOD1

SOD1

SOD1

SOD1

SOD1

SOD1

SOD1

No

rmal

ized

Hu

man

So

d1/

Rat

Pro

lact

in C

op

y N

um

ber

Sod1/Prolactin

Late-responders

Early-responders

Courtesy of Prof. Clive Svendsen, Waisman Center, University of Wisconsin-Madison


QGP-DNA on Frozen Human Tissues

0

200

400

600

800

1000

1200

1400

SMAR

CE1

LSM1

BRF2

FGFR

1

MAPKAPK

ERBB2

PNMT

TOP2A

MYST2

GRB7

Sig

nal

(M

FI)

Normal Breast

Normal Lung

Normal Liver

Frozen human tissues were homogenized with the TissueLyzer (Qiagen) using 5mm stainless steel beads in the 48 sample adaptor and sheared for 2 min. Samples were

centrifuged to remove tissue debris. Approximately 700ug tissue/well was used.

10-plex DNA Panel


QuantiGene® Plex DNA AssayHer2 DNA Copy Number in SKBR3 Breast Cancer Cells

QGP 2.0 - 8-plex/DNA Copy Number Analysis

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

MAPKAPK2

MYST2

BR

F2

FG

FR

1

ER

BB2

GR

B7

PN

MT6

SM

AR

CE1

ch-1 ch-5 ch-8 ch-8 ch-17 ch-17 ch-17 ch-17

Norm

aliz

ed to S

kin F

ibro

bla

st

Normal Skin Fibroblast

MDA-231

SK-BR3

SKBR3/normal Ratio = 6.5

8-Plex Gene Panel:•Normal skin fibroblast

•MDA-231•SKBR3

Her2

In situ HybridizationHer2 amplified in

SKBR3 cells

Her2 = 14Her2 = 2

Ratio of SKBR3/HeLa = 7


Biological Validation of ERBB2 DNA CNV in Breast Cancer Cell Lines

ERBB2 DNA Copy Number in Breast Cancer Cell Lines

0

2

4

6

8

10

12

MCF-7 MDA-MB-231

SK-BR-3 BT-474 UACC-812Fo

ld D

iffe

ren

ce i

n E

RB

B2

Co

py

Nu

mb

er

QG 2.0 Assay – ERBB2 CGH Data

ERBB2


QuantiGene®

Translocation TMPRSS2 - ERG – Fusion RNA in Prostate Cancer

**P <.01*P <.05

**P <.01*P <.05


• QuantiGene 2.0 Reagent System• QuantiGene Plex 2.0 Reagent System

• ViewRNA In Situ Analysis- Single copy mRNA detection in cells- Multiplex detection of mRNAs- Microscope slides & multi-well plates

• QuantiGene Plex 2.0 Reagent System• Procarta Cytokine Plex Kits• Procarta TF Plex Assays• Procarta SH2 Plex Assays

• Stable Cell ines• DeliverX for siRNA• DeliverX for Peptides• Cancer Cell Isolation Kit• Luciferase TF Vectors• Mammalian TF Expression Vectors

• Procarta TF Plex• PD Arrays• Nuclear Extraction Kits• EMSA Kits• Procarta Cytokine Plex Assays• TF ELISA Kits• Cancer Antigen ELISA Kits

Panomics Product Overview

www.panomics.com


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