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
3Affymetrix Confidential
• 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
4Affymetrix Confidential
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
6Affymetrix Confidential
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
7Affymetrix Confidential
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
8Affymetrix Confidential
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
9Affymetrix Confidential
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
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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
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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
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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
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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%
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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)
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Cooperative hybridization
Simple hybridization
May 2006
QuantiGene® 2.0 Assay Diagram
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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®
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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.
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September 2006
MAQC StudyResults Validate the Performance of QuantiGene® Assay
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• 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®
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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
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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.
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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.
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QuantiGene® Plex 2.0 Assay
Bead 1 Bead 2 Bead n
May 2006
>6,000 Panels
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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
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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
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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
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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
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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.
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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.
31Affymetrix Confidential
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
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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.
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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
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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
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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
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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
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QuantiGene® ViewRNAsiRNA Knockdown HPRT- HeLa Cells
HPRT (Green)ACTB (Red)
Nuclei DAPI Stained (Blue)
0nM siRNA 0.04nM siRNA 10nM siRNA
38Affymetrix Confidential
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
39Affymetrix Confidential
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
40Affymetrix Confidential
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
41Affymetrix Confidential
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).
42Affymetrix Confidential
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.
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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.
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Breast Tumor
20XColon Tumor Prostate Tumor
Lung Tumor
QuantiGene® ViewRNAHuman FFPE Archived Tissue Sections - Her2 Expression
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QuantiGene® ViewRNAAnalysis of Her2 Expression in Breast Cancer Progression
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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.
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Breast cancer FFPE tissue section Her-2 / Gill’s Hematoxylin
20X
QuantiGene® ViewRNAHuman Breast Cancer FFPE Tissue Section – Her2 Expression
48Affymetrix Confidential
Breast cancer FFPE tissue section EGFR / Gill’s Hematoxylin
20X
QuantiGene® ViewRNAHuman Breast Cancer FFPE Tissue Section – EGFR Expression
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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.
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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.
51Affymetrix Confidential
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
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QuantiGene® ViewRNAMouse Sagittal Brain Section – Drd1a – Dopamine Receptor D1A
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QuantiGene® ViewRNAMouse Sagittal Brain Section – Dopamine Receptor D1A
QuantiGene® Applications
RNAi Gene Silencing and Compound Screening
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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
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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
• 3’ IVT• WT Gene Plate Array
• 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
• 3’ IVT• WT Gene Plate Array
• miRNAWG Genotyping
• SNP 6.0- association & CN• Cyto chips
• Axiom• DMET chips
57Affymetrix Confidential
•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
58Affymetrix Confidential
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
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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
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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.
61Affymetrix Confidential
September 2005
QuantiGene® Plex Application: siRNA Knock-downEffects of IL-8 siRNA Knockdown on 11 Pathway Genes
62Affymetrix Confidential
QuantiGene® ViewRNATransfection and Untreated HeLa Cell Controls – HPRT
0nM siRNA -Transfected Cells only – No Transfection
HPRT (Green)ACTB (Red)
Nuclei DAPI Stained (Blue)
63Affymetrix Confidential
QuantiGene® ViewRNAsiRNA Knockdown HPRT- HeLa Cells
HPRT (Green)ACTB (Red)
Nuclei DAPI Stained (Blue)
0nM siRNA 0.04nM siRNA 10nM siRNA
64Affymetrix Confidential
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.
65Affymetrix Confidential
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
66Affymetrix Confidential
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
67Affymetrix Confidential
QuantiGene® Plex Tox Screening Application Drug Induction of Transporters & P450 Metabolizing Enzymes
0.0
1.0
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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
QuantiGene® Applications
RNA Quantification from Archival FFPE Samples
69Affymetrix Confidential
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.
70Affymetrix Confidential
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
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25
27
29
1h- 1h+ 16h- 16h+ 1h- 1h+ 16h- 16h+ 1h- 1h+ 16h- 16h+
ACTB PPIB GAPD
Ct
0
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500
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1h+
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-
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+
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1h+
16h
-
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+
1h-
1h+
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-
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
71Affymetrix Confidential
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)
72Affymetrix Confidential
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
73Affymetrix Confidential
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
74Affymetrix Confidential
QuantiGene® FFPE Assay: Quantification of Biologically Relevant mRNAs in 3 & 14 Yr FFPE Samples
0
1
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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
75Affymetrix Confidential
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
76Affymetrix Confidential
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
77Affymetrix Confidential
QuantiGene® Plex FFPE AssayBiomarker Validation - Affymetrix GeneChips to QGP Assays
AffymetrixGeneChip®
PanomicsQuantiGene® Plex
78Affymetrix Confidential
QuantiGene® Plex 2.0 FFPE Assay Her-2 RNA Expression in Breast Cancer FFPE Biopsies
Soonmyung Paik - NEJM March 27, 2008
79Affymetrix Confidential
Reliable Gene Expression Profiling of HumanFFPE Tumor Xenografts
Normalized FFPE Sample Data
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500.0
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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.
80Affymetrix Confidential
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
QuantiGene® Applications
RNA Quantification
from Blood Samples
82Affymetrix Confidential
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
83Affymetrix Confidential
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
.
84Affymetrix Confidential
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%.
85Affymetrix Confidential
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
86Affymetrix Confidential
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
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10000
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Whole Blood
RB
C-l
yse
d B
loo
d
BCL-XL
R2=0.998
1
10
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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
87Affymetrix Confidential
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
88Affymetrix Confidential
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
90Affymetrix Confidential
QuantiGene® Plex DNA Assay Single Copy Gene Resolution Discriminates 0.5 to 6 Copies
0
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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
91Affymetrix Confidential
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
92Affymetrix Confidential
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?
93Affymetrix Confidential
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
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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
94Affymetrix Confidential
QGP-DNA on Frozen Human Tissues
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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
95Affymetrix Confidential
QuantiGene® Plex DNA AssayHer2 DNA Copy Number in SKBR3 Breast Cancer Cells
QGP 2.0 - 8-plex/DNA Copy Number Analysis
0.0
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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
96Affymetrix Confidential
Biological Validation of ERBB2 DNA CNV in Breast Cancer Cell Lines
ERBB2 DNA Copy Number in Breast Cancer Cell Lines
0
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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
97Affymetrix Confidential
QuantiGene®
Translocation TMPRSS2 - ERG – Fusion RNA in Prostate Cancer
**P <.01*P <.05
**P <.01*P <.05
98Affymetrix Confidential
• 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
99Affymetrix Confidential
Thank You