QuantiGene® & Procarta®
Single and Multiplex Solutions for RNA
DNA & Protein Quantification
April 20, 2009
Presentation Topics
ƒ
Panomics Product Overview
ƒ
QuantiGene® bDNA Technology Overview
ƒ
ƒ
ƒ
QuantiGene® Applications
- RNAi
- Compound Screening – GE HTS Plex:Plex screening
- Blood
- Formalin Fixed Paraffin Embedded Tissue (FFPE)
- In situ RNA “ViewRNA”
- DNA Copy Number Variation (CNV)
Procarta® Cytokines, Transcription Factors, & SH2 Domains
Summary
Affymetrix Confidential
2
Panomics Product Overview
• ViewRNA In Situ Analysis
Single copy mRNA detection in cells
• QuantiGene 2.0 Reagent System
• QuantiGene Plex 2.0 Reagent System
•
•
•
•
QuantiGene Plex 2.0 Reagent System
Procarta Cytokine Plex Kits
Procarta TF Plex Assays
Procarta SH2 Plex Assays
• Stable Cell Lines
• DeliverX for siRNA
• DeliverX for Peptides
Affymetrix Confidential
• Procarta TF Plex
• Procarta Cytokine Plex Assays
• TF ELISA Kits
3
QuantiGene® Assay System
Value & Benefits
ƒ
ƒ
ƒ
ƒ
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
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 by
Siemens as “Versant”
- Ideal to validate siRNA knockdown & detect small differences – Dharmacon
validates all siRNAs using QuantiGene
Flexible Assay Formats, Both Single & Multiplex
- Measure anywhere from 1 to 40 targets
- Wide range of applications, including FFPE & blood
Affymetrix Confidential
4
QuantiGene® Assay Workflow
From Sample to Data in a Few Hybridization Steps
Gene Expression Quantification Directly from the Source
Signal Amplification Assay
No RNA & DNA Isolation
No Reverse Transcription
No PCR
Eliminates Unnecessary Steps
Samples
Cells, Tissues, Viruses,
Bacteria, FFPE, Blood, etc.
Affymetrix Confidential
5
QuantiGene® Single Gene/Well – 96 Well Format
Reagent System Components
Component 2
Component 1
Target Specific Probe Set
Coated 96 well Capture Plate
LE
BP
CE
Universal
Capture Probe
QG Probe Set
Capture Well
Component 3
bDNA Signal Amplification Reagents
Amplifier label
Branched DNA
(Amplifier)
Affymetrix Confidential
Alkaline Phosphatase Conjugated
Oligos
6
Substrate
Glow Luminescence
QuantiGene® 2.0 Assay: Animation
> 7000 Assays
Simple hybridization
May 2006
Cooperative hybridization
Affymetrix Confidential
7
QuantiGene® 2.0 Assay
New Amplifier Generates 10X Increase in Sensitivity
NEW QG2.0
QG1.0
AMP
(15X)
Affymetrix Confidential
8
The New QuantiGene® 2.0 Amplifier
PCR Level Sensitivity Using Signal Amplification
RLU
IFNγ IVT
QG 1.0
New QG 2.0
100 copies
3000 copies
IVT (amol)
IL6 IVT
IL-6 spike
recovery
IL-6 Spike
Recovery
IL-6Spike
spikeRecovery
recovery
IL-6
IL6 IVT + cell lysate
recovery
%%Recovery
100.0
RLU
RLU (s-b)
1000.0
10.0
1.0
0.000
0.001
0.010
0.100
0.000
1.000
IVT concentration
(amol/well)
IVT (amol)
Affymetrix Confidential
140%
130%
120%
110%
100%
90%
80%
70%
60%
50%
40%
9
0.001
0.010
IVTIVT
(amol)
0.100
1.000
The New QuantiGene® 2.0 Amplifier
Enables Important Applications
• RNA measurements directly from
crude lysates in difficult samples
(FFPE, blood)
• Multiplex assays from crude
lysates for HTS primary screening
•
Biomarker analysis
FFPE: Theranostic Assays:
Eli Lilly, Pfizer, Gray Lab, Paik Lab, etc.
Gene Expression High
Throughput Screening
Highly Sensitive ViewRNA
In Situ Assays
• Novartis Cambridge early adopter
200,000 data points/day
• Single mRNA detection in cells
Direct from blood RNA assays
Amgen clinical trial, DioGenix
DNA CNV Assays:
Abbott
Affymetrix Confidential
In situ single RNA molecule detection
10
Merck, BD, Roche, Pfizer (3 sites), GSK,
AstraZeneca, TransTech, Solvay, RXi,
Broad/MIT, Harvard
QuantiGene® in MicroArray Quality Control Study
Assay Precision & Accuracy
Assay Precision
Assay precision was determined as % CV’s of
measurements made on 224 RNAs in two reference RNA
samples:
A= Universal Human Reference RNA (Stratagene)
B= Human Brain Total RNA (Ambion).
Assay Accuracy
Assay accuracy was determined as the % difference
between actual and predicted values of 118 common targets
in samples:
C= 75% A + 25% B
D= 25% A + 75% B
¾ QuantiGene outperformed Taqman in terms of assay precision and accuracy.
¾ “The data for the QuantiGene platform were notable given that these values encompass the system-wide
accuracy of the platform.”
Data are from Canales et al. 2006. Nature Biotechnology 24(9): 1115-1122.
Affymetrix Confidential
11
September 2006
September 2006
Luminex® Multiplex Assay Menu
Luminex:
• Multiple Targets
• Multiple Samples
Luminex
ELISA- Multiple Samples
Array – Multiple Targets
•
•
•
•
Affymetrix Confidential
QuantiGene Plex - Gene Expression
Procarta - Cytokines
Procarta - Transcription Factors
Procarta - SH2 Domains
12
QuantiGene Plex® 2.0
Based on Luminex® xMAP Technology
5.6μm & 6.5μm
Microspheres
Polystyrene &
Magnetic
Red & Infared
fluorophores
X MAP
Detection System
The Luminex platform is a robust flow cytometer
based instrument, installed in > 5600
laboratories.
Affymetrix Confidential
Analyte coupled
to microsphere
Red laser - bead classifier
Green laser - reporter quantifier
13
2-Laser
Flow Cytometer
QuantiGene Plex® 2.0
Reagent System Components
Component 1 - Luminex beads
Bead n
Bead 2
Bead 1
Component 2 - QGP 2.0 probe set
Capture Extender Probes
Label Extender Probes
Blocking Probes
NOTE: In addition the assay
kit contains buffers, reagents,
and plates necessary to run
the assay.
Component 3 - Branched DNA Amplifier
Pre-Amplifier
Affymetrix Confidential
Amplifier
Biotin
Labeled
probe
14
Streptavidin-PE
Reporter molecule
QuantiGene® Plex 2.0
Multiplex bDNA Assay Animation
> 1200 Panels
May 2006
Bead 1
Affymetrix Confidential
Bead 2
Bead n
15
QuantiGene® Plex 2.0
Sensitivity and Dynamic Range
100000
CSF2
VEGF
10000
IFNγ
IL10
1000
MFI
IL1β
IL6
100
IL8
TNFα
10
IL2
DAP
1
GAPDH
0.001
0.01
0.01
1
10
IVT RNA (amol)
•
•
•
•
Data for eleven (11) targets in cytokine panel
Dilution series of IVT RNA
Greater than 3 logs linear dynamic range
Sensitivity > 0.01 amol (~1000 copies)
Affymetrix Confidential
16
100
QuantiGene® Plex 2.0
Concordance with QuantiGene® 2.0 Single plex
Inflammatory Response Stimulated by PMA/LPS
QuantiGene® Plex
QuantiGene®
10000
10000
1000
1000
Samples: U937 treated +/- PMA/LPS
Conclusion: Matched Pair Analysis Correlation = 0.94
Affymetrix Confidential
17
IFNG
IL-10
IL-6
TNFa
IL-2
+PMA/LPS
-PMA/LPS
VEGF
IFNG
IL-10
IL-6
TNFa
IL-2
0.1
VEGF
0.1
CSF2
1
GAPD
1
IL-8
10
IL-1B
10
CSF2
100
GAPD
100
IL-1B
IL-8
RLU
100000
MFI
100000
+PMA/LPS
-PMA/LPS
QuantiGene® Assay Specifications
Singleplex
Multiplex
Signal amplification platform:
1.0
2.0
1.0
2.0
Number of Targets per well:
1
1
3-30
3-30
Limit of Detection (RNA copy #):
3000
200
~24,000
1000-2000
Limit of Quantitation (RNA copy #):
6000
500
~60,000
2000-4000
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%
Affymetrix Confidential
18
Applications:
RNAi & Compound Screening
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 siRNA
composition & concentration of SMARTpools®
members.
• No RNA isolation or purification allows high precision
for fine 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
• Dharmacon validates all of their siRNAs using
QuantiGene – thus Panomics has off-the-shelf probe
sets for all of Dharmacon’s siRNAs.
• Other companies that use QuantiGene to validate
their siRNAs – Alnylam (Roche) Germany, Alnylam
Boston, Pfizer, RXi, etc.
Technical Bulletin #006 www.dharmacon.com
Affymetrix Confidential
20
QuantiGene® Plex 2.0
Compound Screening – Cell Lines
High precision and excellent signal/background enable drug screening assays
•
•
•
Normalized to housekeeping gene GAPDH
Affymetrix Confidential
21
HUVEC cells seeded at 10,000 cells/well
Treated with IL-1β at various doses for 24hr
QG® Plex - cell lysates used directly
QuantiGene® Plex
“A rapid, simple method for generating reliable, consistent multiple gene expression data.”
¾ 14 plates using qPCR becomes 1
plate us QG Plex
¾ Tighter data using QG Plex allows
better decision making
Affymetrix Confidential
22
GE HTS Screening
QuantiGene® Plex 2.0 and Luminex® Automated Solution
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ
Runs in 96 or 384 well format on Luminex® platform
Multiplex- ‘Plex in Plex’ strategy (simultaneous plexing of samples
and targets or samples and time points, treatment doses, etc.)
Measures genes direct from lysate with minimal sample handling;
eliminates RNA isolation
Detect low expressed mRNAs with minimal cells; sensitive down
to 1000 transcripts
Low cost per data point
High-throughput and automatable; can generate from ~ 50,000
(96 well plates) to 200,000 (384 well plates) data points/day
using up to 8 X 8 plex reactions/well
Affymetrix Confidential
23
QuantiGene® Plex 2.0 GE HTS Screening
Conventional ‘Linear Workflow’
Plate 1
1
treat cells with
compounds
lyse cells
Plate 2
lyse cells
Plate 3
lyse cells
Plate 4
lyse cells
Plate 5
lyse cells
Plate 6
lyse cells
Plate 7
lyse cells
Plate 8
Affymetrix Confidential
lyse cells
3
4
5 6
7
2
3
4
5 6
7
2
3
4
5 6
7
2
3
4
5 6
7
2
3
4
5 6
7
2
3
4
5 6
7
2
3
4
5 6
7
capture analytes
with beads
24
wash & add label
read-out in
Luminex
Data
wash & add label
read-out in
Luminex
Data
wash & add label
read-out in
Luminex
Data
wash & add label
read-out in
Luminex
Data
wash & add label
read-out in
Luminex
Data
wash & add label
read-out in
Luminex
Data
wash & add label
read-out in
Luminex
Data
wash & add label
read-out in
Luminex
Data
8
8
8
8
8
8
capture analytes
with beads
1
treat cells with
compounds
2
capture analytes
with beads
1
treat cells with
compounds
8
capture analytes
with beads
1
treat cells with
compounds
7
capture analytes
with beads
1
treat cells with
compounds
5 6
capture analytes
with beads
1
treat cells with
compounds
4
capture analytes
with beads
1
treat cells with
compounds
3
capture analytes
with beads
1
treat cells with
compounds
2
8
QuantiGene® 2.0 “Plex in Plex”
GE-HTS Pooling Strategy
Zip code
•
•
8 unique zip codes
64 unique bead colors
5.6µm
or
6.5µm
1
2
3
4
5
6
7
8
Panel 1
9
10
11
12
13
14
15
16
Panel 2
17
18
19
20
21
22
23
24
Panel 3
25
26
27
28
29
30
31
32
Panel 4
33
34
35
36
37
38
39
40
Panel 5
41
42
43
44
45
46
47
48
Panel 6
49
50
51
52
53
54
55
56
Panel 7
57
58
59
60
61
62
63
64
Panel 8
Bead Set 21
Bead Set 16
Affymetrix Confidential
25
QuantiGene® 2.0 “Plex in Plex”
GE-HTS Pooling Strategy
Plate 1, well A1
2
1
4
Plate 2, well A1
3
5
6
7
10
9
12
15
8
17 18
11
13
Plate 3, well A1
14
20
21
23
16
19
22
4
6
5
7
12 13 14
15 16
28
26
27
29
31
30
Plate 5, well A1
33
36
38
40
41
42
43
45
44
47
46
48
Plate 7, well A1
49
52
50
51
53
55
54
56
Plate 8, well A1
57
60
58
59
61
63
62
64
Luminex 384 Well HTS System
Well A1
17 18 19
35
37
39
32
34
Plate 6, well A1
Amplify & Label
20 21 22
23 24
8
25 26 27
28 29 30
25
24
9 10 11
1 2 3
Plate 4, well A1
33 34 35
36 37 38
39 40
31 32
49 50 51
52 53 54
55 56
41 42 43
44 45 46
47 48
57 58 59
60 61 62
63 64
Affymetrix Confidential
High Throughput + High Content Screening
Achieves:
• Increased throughput (8 fold compression into analytical plate)
• Better cost efficiency – more data per detection reagents
• 50,000 data points a day – 96 well format
• 200,000 data points a day – 384 well format
26
QuantiGene® Plex 2.0 “Plex in Plex”
GE HTS – Workflow – 8 x 8 format
Add Compounds
Add Luminex beads
+ probe set
Lyse cells
Incubate cell + compounds
Cell culture plate
Add Pre-Amp and incubate
for 1hr at 50º C
Hybridize overnight
Add Amplifier and
incubate for 1hr at 50º C
Label Probe and
Detection.
Magnet Separation
Twister to reader
After overnight hybridization 8 plates are
combined in less than 30’ to form one
analysis plate. Each well now contains 64
unique beads.
All steps have commercially available automation options
Affymetrix Confidential
27
QuantiGene® Plex 2.0 “Plex in Plex”
Measure 8 Plex in 25K Compound Treatments/ Day
The Luminex HTS system can process a 384 well plate in ~90 minutes or 8 plates a day
Plex
Cell culture
Analytical
plates
plates
per day
per day
Data points
Data points
per
per
analytical
analytical
well
8 Plex
64
8
Data points
per
working day
Days for 1.2
treatments million screen
per day
plate
24,576
64
196,608
•
64 cell culture plates compressed into 8 analytical plates
•
25,000 data points/analytical plate
•
200,000 data points - 8 analytical plates/day
•
25,000 compound treatments/day
•
1.2 million compounds screened in 49 days
Affymetrix Confidential
Compound
28
24,576
49 days
QuantiGene® Plex 2.0 GE HTS
Novartis Validation of QGP Plex:Plex
An example:
Gene expression responses
to active and inactive compounds
Affymetrix Confidential
29
500 Analytes
Affymetrix Confidential
30
Applications:
Blood & Formalin Fixed Paraffin
Embedded (FFPE)
QuantiGene® 2.0 Blood Reagent System
Assay Workflow and Benefits
Whole- & Dried Blood
Blood collected in anticoagulant tubes or as dried spots
PAXgene
Tempus
Blood collected in PAXgene or Tempus tubes
No RNA purification
- Works directly from prot. K digested whole blood, PAXgene®-, or Tempus® tubes
No Reverse transcription or amplification
- Avoids biases inherent to RT & PCR amplification
- Avoids enzyme inhibitors found in blood
No Globin RNA depletion required
July 2006
Multiplex up to 40 genes/well
Small volume of blood required - typically <15 ul
Affymetrix Confidential
32
QuantiGene® 2.0 Blood Reagent System
Insensitive 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.
Affymetrix Confidential
33
QuantiGene® 2.0 Blood Reagent System
Measure Gene Expression From Subpopulations of Whole Blood Samples
120
Quantitative Detection of
Cell Marker Genes in
Heparinized Whole Blood
Using the QuantiGene®
Blood Reagent System.
CD66d (Neutrophil)
CD3E (T cell)
100
CD61 (Platelet)
Net RLU
80
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 targetspecific Probe Sets. The
mean CVs were 6%.
60
40
20
0
0
5
10
15
20
25
Microliter Whole Blood
Affymetrix Confidential
34
30
35
QuantiGene® Blood Reagent System
PAXgene Workflow
PAXgene 2.5ml blood/~9.5ml tube
•Centrifuge 5’ 3000xg
•Resuspend in QG Lysis buffer
+ prot K 60ºC 1h
•Hybridize QG or QGP
Eliminates Unnecessary Steps
Affymetrix Confidential
35
QuantiGene® Blood Reagent System
GAPDH Gene Expression in PAXgene Stabilized Whole Blood
Nucleic acid pellet formed in PAXgene stabilized blood was lysed and assayed
using the QuantiGene Blood Kit.
Affymetrix Confidential
36
QuantiGene® Blood Reagent System
PAXgene and Tempus Workflow
PAXgene 2.5ml blood/~9.5ml tube
Tempus – 3ml blood/~9.5ml tube
54ul = 15ul blood
•Centrifuge 5’ 3000xg
•Resuspend in QG Lysis buffer
+ prot K 60ºC 1h
•Hybridize
•Centrifuge 1h at 5,000xg
•Wash pellet 70% ETOH (H2O)
•Resuspend in 300ul QG Lysis Buffer
•Hybridize 1.5ul = 15ul blood
Affymetrix Confidential
37
QuantiGene® Blood Reagent System
Acute Myeloid Leukemia (AML) Blood – Biomarker “A”
15µl PAXgene- & Tempus treated whole blood
BIRC5 - PAXgene & Tempus
35
30
RLU & S/N
25
20
BIRC 5
15
10
5
0
PAXgene
Affymetrix Confidential
Tempus
Background
38
PAXgene S/N
Tempus S/N
QuantiGene® Plex 2.0 Blood Reagent System
Whole Blood Gene Expression Analysis
-Excellent correlation between
whole blood and RBC-lysed
blood.
-Very good correlation between
whole blood and purified RNA.
BCL2L1
Expected
Expected
slopeslope
8, actual
8, actual
2.3 2.3
RNA extraction
RNA extraction
efficiency:
efficiency:
2.3/8 2.3/8
= 29%= 29%
Slope=2.3378
-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 QuantiGene® Plex.
-Gene List: IL2, TNFα, IL10, IL6, IL1B, IFNγ, IL8,
CSF2,GAPDH, RELB, A20, CDKN1, NFKβ1, NFKβ2,
RELA, NFKβIA, BAK, FASL, FAS, RAFTK, BAD, BCL2,
IL6R, BCL2L1, ACTB and CFLAR.
Affymetrix Confidential
39
QuantiGene® 2.0 FFPE Reagent System
FFPE Gene Expression Profiling Solution with a Simple Assay Workflow
No Chemical Dewaxing Required
Optional
No RNA purification
- Works directly from prot K digested tissue homogenates
No reverse transcription or amplification
- Avoids biases inherent to RT & PCR amplification
Insensitive to chemical modifications of RNA bases
Insensitive to RNA degradation
- Efficient capture of even highly degraded RNA
April 2006
Compatible with H&E stained sections
No chemical dewaxing required
Multiplex up to 40 genes/well
Affymetrix Confidential
40
QuantiGene® FFPE Assay
Quantification is Less Affected by RNA Degradation
0.5kb
0
9
NaOH (minutes)
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
Affymetrix Confidential
41
QuantiGene® 2.0 FFPE Assay
Quantity-Quality Assessments of 3 & 14 Yr Old FFPE Samples
Quantity Assessment
28S/18S = 1.0
28S/18S = 0.25
-18S Ribosomal DNA probe sets are used to
quantify the number of cells in a FFPE sample.
-28S Ribosomal RNA probe sets are to test
the quantity of the RNA in a given FFPE sample.
Quality Assessment
3N 3T
14N 14T
Ratio 28S rRNA/18S rDNA:
-Ratios vary from 0.1-2.0
28S
-Depending on ratio more or less sample is needed to
quantify mRNA expression of equal copy number/cell.
0.1 ratio still gives reliable data.
18S
Agarose Gel
Affymetrix Confidential
42
QuantiGene® 2.0 FFPE Assay
Comparison 3 & 14 yr old FFPE samples – Housekeeping Genes
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
R²=0.9938
28S
18S
Agarose Gel
Affymetrix Confidential
43
QuantiGene® 2.0 FFPE Assay
Comparison 3 & 14 Yr Old FFPE Samples – Target Gene
3N 3T 14N 14T
5
18S
RNA from 3 and 14
year old FFPE
samples of lung
tumor and adjacent
normal tissues run
on an agarose gel.
Fold Change
¾
28S
3T/3N
14T/14N
4
3
2
1
0
LDHA
Agarose Gel
¾
1000
RLU
¾
Raw data for LDHA and RPL32 mRNAs
from dilution series of 3T and 14T FFPE
tissue homogenates, showing linear dose
response.
1
0.1
1
10
100
1000
Sample Input (ng equivalent)
3T LDHA
3T RLP32
Affymetrix Confidential
14T LDHA
14T RLP32
44
RPL32
In agreement with the literature,
QuantiGene® detected 2-fold
induction of LDHA mRNA in tumor
relative to normal tissue even in
highly-degraded 14-yr old sample
(Beer et al. 2002).
100
10
RPL19
QuantiGene® FFPE Assay
Biomarker “A” RNA Expression - Limiting Dilution Experiment
2 - H&E stained
10 - unstained sections
30 Tissue Strips (6 x 2 = 12mm² x 5µm)
A
6mm
6 mm
6mm
2 mm
B
C
12mm²
Section A: 5 strips
Section B: 4 strips
Section C: 3,2,1 strip(s)
Human Prostate Tumor
Biomarker A - Dilution Series - 1-5 Strips in duplicate - RLU - LMax
120
100
RLU
80
EIF4E
60
40
20
0
sample
1a
sample
1b
sample
2a
sample
2b
sample
3a
sample
3b
sample
4a
sample
4b
sample
5a
sample
5b
Bkg
-5 tissue strips = 60mm² x 25µm
-4 tissue strips = 48mm² x 20µm
-3 tissue strips = 36mm² x 15µm
-2 tissue strips = 24mm² x 10µm
-1 tissue strip = 12mm² x 5µm
-All tissue strips homogenized in 300µl buffer + prot K
Prostate Tumor (1-5 Strips in duplicate)
-Biomarker A’s gene expression is constant across 1- 5 tissue strips and for both biological replicates
-One tissue strip is 30X > background
-Overall all CVs = 3.2%
Affymetrix Confidential
45
All tested
in duplicate
QuantiGene® FFPE Assay
Quantify RNA Expression Directly from H&E Stained Sections
Biomarker “A” + Housekeeping Genes (ACTB, RPS20, RPL32)
Comparison Same Prostate Tumor Stained, Unstained & Stained ETOH precipitated
600
Stained
Unstained
ETOH Precipitated
500
RLU
400
ACTB (3ul)
RPS20 (6ul)
300
Biomarker "A" (40ul)
RPL32 (10ul)
200
100
0
Sample A
(stained slides,
THS)
Sample B
(unstained slides,
THS)
Sample C
(Percipitated
sample A)
• Prostate tumor - 5 sections (6x6mm-5µm) of stained or unstained - homogenized in 300µl buffer + prot K.
• H&E stain did not inhibit the QuantiGene measurements of all 4 genes
and is comparable to unstained sections.
Affymetrix Confidential
46
QuantiGene® Plex FFPE Assay
Quantify RNA Expression Directly from H&E Stained Sections
Liver FFPE Sections
Day 1 – Section 1 vs. Section 2
Day 1 - 23-Plex - 20 Target Genes & 3 HKGs - Background Subtracted (MFI)
Section1
Section 2
1800
1600
36mm²
5µm thick
1400
6mm
6mm
6mm
6mm
M FI
1200
1000
Day 1 - Section 1
800
Day 1 - Section 2
600
400
200
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Genes
Day 2 – Section 1 vs. Section 2
-Sections 1 & 2 - each 6mm x 6mm x 5µm
Day 2 - 23 Plex - 20 Target Genes & 3 HKGs - Background Subtracted (MFI)
1800
-Each section homogenized in 300µl buffer + prot k
1600
1400
- Day 1 and Day 2 – Section 1 & Section 2 compared
using 23 gene plex panel
M FI
1200
1000
Day 2 - Section 1
800
Day 2 - Section 2
600
-Homogeneous expression of both sections for all 23
genes
400
200
0
1
-Overall 8.6% CVs
Affymetrix Confidential
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Genes
47
QuantiGene® 2.0 FFPE Assay
Measurement of a Cancer Gene Panel in FFPE Prostate Tumors
¾ Macro dissected FFPE samples from matched tumor and normal
tissues homogenized in Tissue Homogenization Solution (THS).
¾ 14 prostate cancer gene panel measured in tumor and adjacent
normal tissues.
¾ Gene expression normalized to housekeeping gene RPL19.
¾ The ratio between tumor and normal tissues is calculated and shown
in a 5-tiered categorical scale.
“In macrodissected 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
Affymetrix Confidential
48
QuantiGene® Plex 2.0 FFPE Assay
HER2 RNA Expression in Breast Cancer FFPE Clinical Biopsies
HER2 expression is normalized to the GAPDH, RPLO, TFRC, GUSB, ACTB
Soonmyung Paik - NEJM March 27, 2008
Affymetrix Confidential
49
QuantiGene® FFPE Assay System
Sample Types and Typical Amounts of Starting Material
Cell Number
RNA Quality
RNA Expression
Number of Assays
•~20K cells
• 28S/18S = 1.0
•~10-15 RNA copies/cell
• 300 assays if QG 2.0
Affymetrix Confidential
50
QuantiGene® FFPE Assay System
Controls Reagents
QuantiGene FFPE Proficiency Kit:
• FFPE sections provided with controlled experimental outcome
• Kit includes quantity, quality and normalization controls
SAMPLE ASSESSMENT DATA
9
120
8
Multiple Housekeeper Genes Probe Controls:
• Enables “geometric mean” normalization for improved data analysis
• Large panel to fit needs of individual tissue types
Affymetrix Confidential
51
5
4
40
3
2
20
1
SPT
SCT
SPN
SCN
2029T
2027T
2029N
650T
0
2027N
0
28S RNA RLU
6
60
650N
• 18S Ribosomal DNA Assay—measures sample quantity
• 28S Ribosomal RNA Assay—indicates sample RNA quality
7
80
103N
Sample Quantity and RNA Quality Controls:
18S DNA RLU
100
28S RNA
18S DNA
QuantiGene® 2.0 Assay Translational Medicine Application
Prostate Cancer Translocation: TMPRSS2 & ERG Gene Expression
TMPRSS2
ERG
Vcap-Prostate RNA
6000
5000
RLU
RLU
4000
3000
Vcap-Prostate RNA
2000
1000
0
5ng
2.5ng
1.25ng
0.625ng
0.3125ng
TMPRSS2- ERG
0ng
Vcap RNA
Vcap-Prostate RNA
25
Vcap (ng) + Normal Prostate (ng)
S/N
Signal to Noise
20
15
Vcap-Prostate RNA
10
5
0
5ng
2.5ng
1.25ng
0.625ng
0.3125ng
0ng
RNA
Collaboration with Prof. Martin Sanda, Harvard University
Affymetrix Confidential
52
1.)
2.)
3.)
4.)
5.)
6.)
5
2.5
1.25
0.625
0.325
0
+
+
+
+
+
+
0
2.5
3.75
4.375
4.675
5
= Total RNA (ng)
=
=
=
=
=
=
5
5
5
5
5
5
Applications:
In Situ RNA – QuantiGene® ViewRNA
QuantiGene® ViewRNA Assay
Specifications
Limit of Detection
Plex Level
Assay Format
Detection
Instrumentation
Affymetrix Confidential
Adherent Cells (Plates)
FFPE Tissue (Slides)
1 mRNA copy/cell (TYPE 1)
>1,000 mRNA copies/cell (TYPE 2)
1-10 mRNA copies/cell (TYPE
1)
2 (TYPE 1 + TYPE 2)
1
96- or 384-well plates
Slides
Fluorescent
Chromogenic
Imaging System or
Epi-Fluorescent Microscope
Brightfield Microscope
54
QuantiGene® ViewRNA
Multiplex Analysis of Gene Expression in Single Cells
Total 5hr
• Fluorescent in situ hybridization or chromogenic (FISH or CISH)
• Single copy mRNA detection in single cells
• Multiplex mRNA detection – 5 plex available Q4/09
• Excellent signal-to-noise ratio
• Fast and automatable
Affymetrix Confidential
55
QuantiGene® 2.0 Assay
New Amplifier Generates 10X Increase in Sensitivity
NEW QG2.0
QG1.0
AMP
(15X)
Affymetrix Confidential
56
QuantiGene® ViewRNA
Assay Validation – 18S rRNA
Simultaneous Signal Amplification and Background Reduction
10 μm
16X AMP
Z
Left set
Z
Affymetrix Confidential
1X AMP
Z
18S sense probe
Right set
Z
57
QuantiGene® ViewRNA
HER2 & IL-8 DNA In Situ Assay – HeLa & SKBR3 Cells
Detection in Chromosomal DNA of Single Cells
HeLa
SKBR3
Resolution down to 1KB
HER2/IL-8/DAPI
Affymetrix Confidential
58
QuantiGene® ViewRNA
HER2 mRNA & 18S rRNA In Situ – HeLa & SKBR3 Cells
HeLa
SKBR3
HER2/18S rRNA/DAPI
Assay
ViewRNA™
HER2-C/18S rRNA/DAPI
QG® 2.0
Affymetrix Confidential
59
AV. copy # /cell
5.4
6.4 ± 2.3
QuantiGene® ViewRNA
Transcriptional mRNA Heterogeneity
• Time course of PMA induction of IL-6 & IL-8 in HeLa cells
• Induction kinetics at the single cell level
0 hr
0.5 hr
1 hr
2 hr
IL-8 / 18S / DAPI
IL-6 / 18S / DAPI
Affymetrix Confidential
60
4 hr
8 hr
QuantiGene® ViewRNA
Multiplex mRNA Detection
IL-6 & IL-8 mRNA Detection – Single Cell Multiplex Detection
IL-6 (green) & IL-8 (red) in PMA induced HeLa Cells
DAPI
IL-6 & IL-8
IL-6
IL-8
Affymetrix Confidential
61
QuantiGene® ViewRNA
Detection of HCV RNA
Huh7 - HCV Replicon
Huh7 + HCV Replicon
40X
18S in Red HCV in Green
64X
Affymetrix Confidential
62
QuantiGene® ViewRNA
Automation of HTS 96/384 plates
5 Hours from Fixation to Image Analysis
Treat cells
Amplify & label cells
Permealize cells
Fix cells*
DAPI stain cells*
*Stop assay (optional)
Affymetrix Confidential
Hybridize cells with LEs*
Scan plate
63
Image Analysis
QuantiGene® ViewRNA
Quantification of IL-8 Expression in PMA induced HeLa Cells
0.12ng PMA/well
3.33ng PMA/well
0.37ng PMA/well
1.11ng PMA/well
10ng PMA/well
PMA Induction of IL-8 in HeLa Cells
Medean RNA dot counts/cell
41
36
31
26
21
16
11
6
1
0.01
0.10
1.00
PMA (Log of ng/well)
Dose Response
PMA Induction of IL-8 Transcription in HeLa Cells
HeLa cells cultured on the poly-lysine coated glass bottom 384-well plate,
treated with PMA for 4hrs and processed. The images were acquired
using ArrayWoRx MF (Applied Precision) and analyzed using CyteSeer
ViewRNA Software (Vala Sciences).
Affymetrix Confidential
64
10.00
QuantiGene® ViewRNA
Chemiluminescence – Reporter Gene Assay Format
Chemiluminescence – LMax Luminometer
400X amplifier
QGProbe-AP
ViewRNA - AP Label
Label
+ Probe
Alkaline phoshatase conjugated oligos
AP Chemiluminescent
100
Substrate
80
RLU
60
40
Substrate
Glow Luminescence
20
1
K
PG
IB
PP
T
H
PR
2
H
ER
8S
en
-1
Well
N
eg
se
e
pr
ob
RNA
N
eg
o
-n
N
eg
-H
C
V
0
Above - chemiluminescence signal from entire well of a 384-well plate (LMax Luimiometer) – HeLa cells hybridized with multiple
QG probe sets & compared to QG ViewRNA measuring signals in individual cells using fluorophores (below).
Fluorescence – ArrayWoRx High Content Imager
No Probe
Her2
Affymetrix Confidential
HPRT
PPIB
65
PGK1
QuantiGene® ViewRNA
Mouse C2C12 Myoblast Cell Differentiation to Myotubes
T=2
T=0
T=5
Bright field images of C2C12
differentiation at T = 0, 2 and
5 days in 96 well plate.
Affymetrix Confidential
66
QuantiGene® ViewRNA
Increased Expression of Myogenin Expression in C2C12 Myotubes
T=0
T=2
Myotubes expressing myogenin
C2C12 cells cultured in 96 well plate
were formaldehyde fixed at 0, 2 and 5
days differentiation. The fixed cells
were permeabilized and probed for
Myogenin expression using QG
ViewRNA assay. (Blue = DAPI nuclear
stain, Green = Myogenin mRNA)
Affymetrix Confidential
T=5
67
Myotubes expressing myogenin
T=5
Myotubes
expressing
myogenin
Affymetrix Confidential
68
QuantiGene® ViewRNA
FFPE Archived Tissue Sections - Her2 Expression
Breast Tumor
Lung Tumor
Colon Tumor
Prostate Tumor
Affymetrix Confidential
69
20X
Breast Cancer Tissue
Adjacent tissue 1.5 cm away from tumor
Her2 / Hematoxylin
Her2 / Hematoxylin
Tumor
20X
Her 2 (ERBB2) mRNA in Breast Cancer FFPE sections was labeled using QuantiGene ViewRNA 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.
Affymetrix Confidential
70
Detection of Differential Her2 Expression
Using FFPE ViewRNA
Low
Differential Her2 expression
detected in 2 cases of breast
cancer tissues using QuantiGene
ViewRNA for FFPE with Her2
probe.
Left: image of breast cancer
tissue expressing high level of
Her2 in the cancer cells .
Right: image of second breast
cancer tissue showing low/no
expression of Her2 in the cells.
Bottom: QG2.0 analysis confirms
the high (left) and low expression
(right) of Her2 in the tissues.
Her2 (Bright field)
high
25
QG2.0 Her2 /18S expression in the FFPE tissues
H e r2 /1 8 S
20
15
10
5
0
A5
Affymetrix Confidential
C6
71
Comparison of FFPE ViewRNA sensitivity
to radioactive probe
Arany et al.
Journal of Clinical Endocrinology and Metabolism
Vol.79 No. 6, p 1871
FFPE ViewRNA
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
chromogenic substrate incubation.
Affymetrix Confidential
72
In situ RNA detection in FFPE samples
CK19 (Cytokaretin 19) is an epithelial cell specific gene thus can be used to identify
epithelial cells in a mixture of cells such as in tissues.
20X
Bright field
CK19
Adjacent
normal
tissue
In the normal tissues,
the exclusive staining
of the ck19 to the
epithelium indicates
the appearance of
healthy ductal
structures.
In the breast cancer
tissue, ck19 probes label
cells that are not
organized into ductal
structure, indicating the
advanced cancerous
stage in which the
cancer cells (epithelial
origin) have infiltrated
the surrounding tissues.
Breast
cancer
tissue
(Grade III
Stage IV)
Hematoxylin stained
Affymetrix Confidential
Fluorescent images
73
Probe Sets Demonstrating Variable Expression
Levels in Tissues – Breast & Colon
Affymetrix Confidential
74
Probe Sets Demonstrating Variable Expression
Levels in Tissues – Liver & Lung
Affymetrix Confidential
75
Probe Sets Demonstrating Variable Expression
Levels in Tissues – Prostate
Affymetrix Confidential
76
QuantiGene® ViewRNA
Power of in situ multiplexing
Q4/09 – Detect 5 RNAs !
Affymetrix Confidential
77
Applications:
DNA Copy Number
QuantiGene® 2.0 DNA Assay
DNA – Plant Zygosity Testing
CryF1 Gene from GMO Corn Leaf
Q u a n t iG en e
®
Zygosit y Da t a : P u rified DN A
Relative Light Units
1.4
1.2
Homozygous
1
0 .8
Hemizygous
0 .6
0 .4
0 .2
Null
0
A5
B3
C1
C 11
D9
E7
S a m p le W ell
Collaboration with Dow AgroSciences
Affymetrix Confidential
79
F5
G3
H1
QuantiGene® 2.0 DNA Assay
Discriminate 0.5 to 6 Copies
• FGFR1 BAC DNA is spiked into mouse genomic DNA
• QG DNA can discriminate from 0.5 to 6.0 copies
y = 8.1274x + 1.2826
R 2 = 0.9957
70
60
RLU
50
40
30
20
10
0
0
1
2
3
4
5
6
7
8
Copy /cell
ƒ
Increasing copies of Human BAC Fibroblast Growth Factor Receptor 1 (FGFR1) DNA (1.3 pg to
18.2 pg) are spiked into a constant amount of mouse genomic DNA (180 ng or 30,000 cell
equivalents, based on 6 pg mouse DNA/cell). The copy number of FGFR1 is quantified using
QuantiGene 2.0 Probe Set specific to the human FGFR1 receptor DNA. 0.5 to 6 copies of
human FGFR1 gene per cell can easily be distinguished.
Affymetrix Confidential
80
QuantiGene® 2.0 DNA Assay
Sensitivity & Dynamic Range of HER2 DNA
HER2
60000
R2 = 0.9982
50000
RL U
40000
30000
20000
10000
0
0
500
1000
MCF-7 Total DNA (ng)
Affymetrix Confidential
81
1500
2000
QuantiGene® 2.0 DNA Assay
Copy Number Variation (CNV) - Biological Validation with Breast Cancer Cell Lines
CGH Data
QG 2.0 Assay – Her2 (v-erb-b2)
F o l d D i ffe r e n c e i n E R B B 2
Co py Nu m b er
ERBB2 DNA Copy Number in Breast Cancer Cell
Lines
12
10
8
6
4
2
0
MCF-7
MDA-MB231
SK-BR-3
BT-474
UACC-812
HER2 (v-erb-b2)
Affymetrix Confidential
82
QuantiGene® 2.0 DNA Assay
Profiling Amplifications of Chromosome 17 in Breast Cancer Cell Lines
A. Selected targets from the Chr. 17q12-q21 region
ID
A
B
C
D
E
F
G
H
I
J
K
Gene
C17orf63
FLOT2
LOC90110
PPARBP
STARD3
ERBB2
GRB7
THRAP4
TOP2A
SMARCE1
D17S800
start on chr17
24112988
24233016
34662949
34818787
35068264
35108000
35154864
35431521
35810481
36040027
36309790
B. Copy number analysis by QG
A
C17orf63
1
MCF-7
0.71
MDA-MB-231
0.62
SK-BR-3
0.78
BT-474
1.57
UACC-812
B
FLOT2
1
0.85
0.70
0.90
1.81
end on chr17
24113525
24233453
34663397
34819235
35067844
35139000
35154438
35431802
35810928
36040599
36310238
Her2
C
D
E
LOC90110 PPARBP STARD3
1
1
1
1.31
1.24
1.00
5.91
4.06
5.04
11.09
7.05
10.59
8.85
3.78
9.98
F
ERBB2
1
1.68
3.70
7.89
10.09
G
GRB7
1
1.86
2.22
5.60
6.16
H
THRAP4
1
1.68
1.28
4.51
6.79
I
TOP2A
1
0.97
1.11
0.85
2.25
J
K
SMARCE1 D17S800
1
1
0.81
0.85
1.43
1.37
0.75
0.88
1.73
0.52
¾Fold change in gene copy number relative to MCF-7 cells are shown.
¾Excellent agreement with FISH data in SKY database for these cell lines.
Affymetrix Confidential
83
QuantiGene® 2.0 Assay
Prostate Cancer Translocation-Gene Fusion
TMPRSS2-ERG Gene Fusion – VCaP cell RNA expression
TMPRSS2
Vcap-Prostate RNA
ERG
6000
5000
RLU
RLU
4000
3000
Vcap-Prostate RNA
2000
1000
0
5ng
2.5ng
1.25ng
0.625ng
0.3125ng
TMPRSS2- ERG
0ng
Vcap RNA
Vcap-Prostate RNA
25
Vcap (ng) + Normal Prostate (ng)
S/N
Signal to Noise
20
15
Vcap-Prostate RNA
10
5
0
5ng
2.5ng
1.25ng
0.625ng
0.3125ng
0ng
RNA
Lu, et al, Urology 2009 in press
Affymetrix Confidential
84
1.)
2.)
3.)
4.)
5.)
6.)
5
2.5
1.25
0.625
0.325
0
+
+
+
+
+
+
0
2.5
3.75
4.375
4.675
5
= Total RNA (ng)
=
=
=
=
=
=
5
5
5
5
5
5
Summary
ƒ
bDNA technology is the basis of FDA-approved viral load assays (HIV and HCV)
and widely adopted in biological research, drug discovery & clinical trials.
ƒ
Proven performance in FDA-commissioned MAQC study demonstrating excellent
correlation with qPCR & microarray platforms.
ƒ
Easy Workflow: QuantiGene® & QuantiGene® Plex work directly from lysates.
ƒ
QuantiGene® Plex measures housekeeping and target genes in the same well for
minimal sample handling & optimal normalization.
ƒ
Numerous applications using QuantiGene® & QuantiGene® Plex including: in situ
RNA-DNA, DNA CNV, Gene Expression HTS – Plex:Plex.
ƒ
Total solution providing sensitive, accurate & precise measurements of genes
from FFPE & whole blood samples enabling retrospective & prospective studies.
ƒ
QuantiGene® ViewRNA enables measurement of single RNA and DNA molecules
in individual cells in a multiplex format.
ƒ
QuantiGene® is linear a assay that easily differentiates 1, 2, and 3 DNA copies.
Affymetrix Confidential
85
Procarta® Multiplex Protein Luminex® -Based Assays
Installed base of > 5,600 instruments
systems at all major pharma, biopharma and
most academic institutes)
We provide the widest menu of reagents for multiplex protein analysis –
a market that is growing rapidly:
Cytokine Profiling Kits
Affymetrix Confidential
Transcription Factor Assay Kits
86
Protein Interaction Domain Kits
Applications for Multiplex Cytokine
•TH1/TH2 Profiling
•Inflammation
•Acute phase proteins
•Disease panels
•Biomarker Discovery/Validation
•Cancer Marker Profiling
•Immune monitoring
•ADME/TOX
Affymetrix Confidential
87
Procarta® Cytokine Kits
Overview
•
More content, more flexible (configured panels, delivered in <1 week)
•
Optimized for sample types: Supernatant, serum, plasma, tissues and other body fluids
•
Assay buffer is species specific: Human, Mouse, Rat, Monkey, Pig
•
Easy to use - No sample dilutions needed, all reagents ready to use at 1X.
•
Standards included
•
Rapid custom target addition program
•
Equal or better specs to competition, low background with better assay range
•
Certificate of analysis available for every order
•
Highly competitive pricing
Affymetrix Confidential
88
Procarta® Cytokine Assay
Workflow
Three simple assay steps and then detect using a Luminex® platform
The Procarta assays are bead based sandwich immunoassays
based on xMAP® detection technology developed by Luminex.
Affymetrix Confidential
89
Procarta® Cytokine Assay
Largest Number of Available Assays
ƒ
ƒ
ƒ
ƒ
ƒ
Human
Non-Human Primate
Mouse
Rat
Porcine
Total
Affymetrix Confidential
73
43
34
27
6
182
90
Procarta® Cytokine Assay
73 Human Cytokine Assays
IL-1 beta
IL-12 (p70)
FGF basic
PDGFBB
IL-1 alpha
IL-13
GCSF
RANTES
IL-1 ra
IL-15
GM-CSF
TGF beta**
IL-2
IL-17A
GRO alpha
TFN alpha
IL-4
IL-17F
Leptin
TNF beta
IL-5
IL-23
MCP-1
MMP-1
IL-6
INF gamma
MCP-3
MMP-2
IL-7
IP10
MIG
MMP-3
IL-8
CRP**
MIP-1 alpha
MMP-9
IL-10
ENA-78
MIP-1 beta
MMP-13
IL-12 (p40)
Eotaxin
NGF
Ghrelin
Resistin
ICAM
PAI-1
IL-31
VCAM
IFN alpha
IFN-omega
MIF
IL-18
Eotaxin 2
SAA
MIP-3 alpha
Eotaxin 3
MMP-12
TRAIL
IFN-beta
HGF
IL-20
ITAC
EGF
IL-21
PIGF
MMP-7
IL-22
E-selectin
M-CSF
TPO
sCD40 ligand
Adiponectin
Affymetrix Confidential
91
43 Cytokines
Specifications
LOD <1 pg/ml
LOQ < 10 pg/ml
Dynamic Range > 2 logs
Mix and Match up to 30
** Should be done as single
New: 30 cytokines
and growing
Procarta® Cytokine Assay
43 Non-Human Primate Cytokine Assays
23 New Targets
IL-8
Granzyme B
IL-6
IL-17A
MCP-3
GM-CSF
IL-1 beta
IL-10
IL-23
MIP-1 alpha
Eotaxin
IL-2
IL-12p40
IFN gamma
MIP-1 beta
IL-4
IL-12p70
IP10
RANTES
IL-5
IL-13
MCP-1
TNF alpha
G-CSF
FGF-Basic
VEGF
TGF beta1
TNF beta
NGF
PDGF-BB
IL-1RA
IL-15
IL-17F
BLC
MIG
Adiponectin
TRAIL
Specifications
LOD <1 pg/ml
LOQ < 19 pg/ml
Dynamic Range > 2 logs
Mix and Match up to 25
sCD40 Ligand
Resistin
API-1
VCAM
ITAC
Affymetrix Confidential
92
MIF
Procarta® Cytokine Assay
34 Mouse Cytokine Assays
Eotaxin
IL-4
IL-6
IP-10
G-CSF
IL-5
IL-9
KC
5 New Targets
BTC
Leptin
GM-CSF
MCP-1
IL-10
IFN gamma
MCP-3
IL-12p40
IL-1 alpha
MIP-1-alpha
IL-12p70
sRANKL
Adiponectin
MIP-2
IFN alpha*
* 2 plex assay kit
IL-1 beta
RANTES
IL13
IFN beta*
IL-2
TNF-alpha
IL-17
TGF beta**
IL-3
VEGF
IL-23
Affymetrix Confidential
93
** Should be run as single
Specifications
LOD <1 pg/ml
LOQ < 19 pg/ml
Dynamic Range > 2 logs
Mix and Match up to 25
Procarta® Cytokine Assay
27 Rat Cytokine Assays
Eotaxin
IL-1 alpha
IL-12p40
NGF
G-CSF
IL-1 beta
KC
RANTES
GM-CSF
IL-4
MCP-1
TGF beta**
6 New Targets
IL-5
IL-17
Leptin
sRANKL
ICAM
IFN gamma
IL-6
IL-10
MCP-3
TNF alpha
MIP-1 alpha
VCAM
IL-18
IL-2
** Should be run as single
VEGF
Specifications
LOD <1 pg/ml
LOQ < 19 pg/ml
Dynamic Range > 2 logs
Mix and Match up to 25
Affymetrix Confidential
94
Procarta® Cytokine Assay
6 Porcine Cytokine Assays
IL-1 beta
IL-6
IL-8
IL-10
TNF alpha
IL-4
Specifications
LOD <1 pg/ml
LOQ < 19 pg/ml
Dynamic Range > 2 logs
Mix and Match up to 25
Affymetrix Confidential
95
Procarta™ Cytokine Assay
Cross-reactivity study
File Name: S:\LeslieS\IFN_corssreactivity_03232009.rbx
Acquisition Date: 23-Mar-2009, 03:32 PM
Reader Serial Number: LX10003077012
RP1 PMT (Volts): 560.68
RP1 Target: 3415
Plate ID:
Well Type Description
A11 X5
IFNalphaAg_dAbMix
B11 X6
C11 X7
D11 X8
IFN-alpha (48) IFN beta (30)IFN OMEGA (68)
10191
4
6
IFNbetaAg_dAbMix
5
3821
16
IFNOmegaAg_dAbMix
Blank
4
5
5
4
11065
7
Any target developed for Procarta panels goes through intense
validation including cross-reactivity studies.
For example, the subfamily of IFN has high homology in protein
sequence, however still very specific antibody pair are found for
each target.
Affymetrix Confidential
96
Procarta® Tissue/Cell Lysate Kits:
Unique Assays for Detection of Intracellular Cytokines
Homogenize
Centrifuge
Analyze
Luminex
Tissues or Cells
•~10 mg in 150µl Procarta lysis buffer
•Centrifuge 14,000rpm
10 min at 4oC
•Dounce 30-40 strokes
•Collect supernatant
•Analyze and/or store at -80ºC
Affymetrix Confidential
97
Procarta® Tissue/Cell Lysate Kits:
Unique Assays for Detection of Intracellular Cytokines
Procarta vs. Linco Plex (Millipore): Rat Brain Tissue Lysates
Procarta cytokine lysis buffer does not damage or denature proteins generating superior
detection signals from cell- or tissue lysates.
Data kindly provided by Japanese customer
Affymetrix Confidential
98
Procarta® and QuantiGene® Plex
Quantify Protein & mRNA from the Same Sample
Use same lysates for both Procarta and QuantiGene Plex:
a.
b.
c.
Supernatant for secreted cytokines - Procarta
Step 1: Procarta lysis buffer – Intracellular cytokines - Procarta
Step 2: Take half of the Procarta lysate + protease K – QuantiGene Plex
Supernatant - secreted cytokines - Procarta
Half sample + protease K - QuantiGene Plex – mRNA
Cell Pellet - Procarta lysis buffer
Affymetrix Confidential
Half sample - Procarta intracellular cytokines
99
Procarta® and QuantiGene®
Quantify Cytokine Protein & mRNA From The Same Sample
U937 Cells induced with LPS
IL-6 Protein & mRNA Expression in
LPS-induced U937 cells
300
250
500
200
400
150
300
100
200
50
100
0
-100
5 10 20 30 1 h 2 h 4 h 8 h 16
min min min min
h
24 48
h
h
40
20000
Protein IL-1B
mRNA IL-1B
30
15000
20
10000
10
5000
0
0
0
50
0
0
-50
-20
TNFa Protein & mRNA Expression in
LPS-induced U937 cells
800
70
3500
700
60
3000
600
50
500
40
400
30
300
20
200
10
100
0
2500
20000
2000
15000
1500
10000
1000
5000
500
0
mRNA IL-8
0
0
0
-5000
Protein IL-8
Relative mRNA Expression
4000
Relative Protein expression
Relative mRNA Expression
25000
5 10 20 30 1 h 2 h 4 h 8 h 16 24 48
min min min min
h
h
h
-500
-100
+ LPS
Affymetrix Confidential
mRNA IL-6
+ LPS
IL-8 Protein & mRNA Expression in
LPS-induced U937 cells
30000
Protein IL-6
-10
24 48
h
h
-5000
+ LPS
35000
5 10 20 30 1 h 2 h 4 h 8 h 16
min min min min
h
-10
0
5 10 20 30 1 h 2 h 4 h 8 h 16
min min min min
h
+ LPS
100
24 48
h
h
-20
Relative Protein expression
600
25000
Relative Protein expression
350
700
Relative mRNA Expression
800
Relative Protein expression
Relative mRNA Expression
IL-1b Protein & mRNA Expression in
LPS-induced U937 cells
Protein TNFa
mRNA TNFa
Procarta® Transcription Factor Assay
Workflow
ƒ
High throughput multiplex EMSA Gel-Shift Assay
ƒ
Assay many samples and many TFs simultaneously
ƒ
ƒ
ƒ
ƒ
ƒ
Affymetrix Confidential
40 plex - and 43 plex panels for a total of 83
Luminex assays
Quantitate TF binding activity from whole cell
lysates (500ng protein) or nuclear extracts (250ng
protein)
Complete assay in 5 hours
Numerous applications; e.g. monitoring disease
states and pathways
Pinpoint TF changes corresponding to compounds
inhibiting receptors, kinases, or other signaling
proteins
101
43-plex
0
Affymetrix Confidential
102
RUNX/AML (44)
AP1-1 (35)
AP2-1 (21)
AR (33)
AT F 2 (26)
NF -Y (47)
CEBP (46)
F AST 1 (12)
C-MYB (51)
CREB (52)
E2F 1-1 (36)
EG R (24)
ELK1 (7)
ER (53)
ET S/PEA (34)
F KH R -1 (56)
G AT A-1 (61)
G R /PR (54)
HIF -1(2) (55)
H N F 1 (64)
IRF 1 (62)
ISRE (28)
MEF -2 (73)
MYO D (37)
NF -1 (27)
N F AT C (76)
NF -E1(YY1) (25)
N F -E2 (20)
NF KB1 (11)
O CT (17)
P53 (18)
PPAR (42)
PAX3 (19)
SMAD (43)
ST AT 1 (63)
ST AT 3 (41)
ST AT 4 (65)
ST AT 5 (66)
NKX-2.5 (32)
T F IID (45)
Procarta® Transcription Factor Assay
Components & Specificity
40-plex
RUNX/AML
30000
AP1
AP2
AR
25000
ATF2
NF-Y
CEBP 10 nM
MYC 10 nM
20000
c-myb
CREB
15000
E2F1
EGR
Elk1
ER
10000
ETS/PEA
T FI I D
FKHR
5000
Smad
ST AT 4
GATA
NF- E2
p5 3
I SRE
NF- 1
FKHR
CREB
AR
GR/PR
HIF-1
Elk1
HI F- 1
HNF1
CEBP 10 n M
IRF1
RUNX/ AM L
ISRE
MEF-2
MyoD
NF-1
NFATc
NF-E1(YY1)
NF-E2
¾ Procarta “Pre-formatted”
Transcription Factor Assay
or
¾ Custom Panels “on-demand”
Procarta® Transcription Factor Assay
Transcription Factor Activity Profiling Assay
HeLa cell nuclear extract +/- PMA
NF-E2
AP-1
NF-1
STAT-4
40 plex TF Activity Results Confirmed by EMSA
Affymetrix Confidential
103
Procarta® SH2 Domain Plex Profiling System
ƒ
ƒ
ƒ
ƒ
ƒ
Luminex Based Assay to measure profiles
the activation of phosphoproteins to
panel of SH2 Domains.
Profile up to 30 different domains from a
single cell lysate.
Complete kit includes all of the reagents
necessary to perform assay.
Simple ELISA-like workflow makes assay
simple and easy to use with data in under
4 hours.
Mix and match to create own unique Plex
Set from the 30 SH2 Domains
Affymetrix Confidential
104
Why SH2 Domains are Important
ƒ
ƒ
ƒ
ƒ
SH2 domains are critical components of the signal
transduction pathways that play a major role in cell
differentiation, proliferation, death and survival.
Useful for studying receptor tyrosine kinases as a
model to manipulate the signal transduction
mechanisms.
Intracellular drug targets can be directed at specific
SH2s to block specific pathways causing a disease
state.
SH2’s can play a potential role as a biomarker in
disease association studies
Affymetrix Confidential
105
SH2 Bind to Receptor Tyrosine Kinases
Affymetrix Confidential
106
Procarta® SH2 Domain Plex
Assay Format - Workflow
1
4
2
5
3
1
Affymetrix Confidential
107
Procarta® SH2 Domain Plex
Example of SH2 Recruitment by Receptor Tyrosine Kinases
EGF
ƒ
ƒ
ƒ
ƒ
Affymetrix Confidential
108
Epidermal Growth Factor
(EGF) binds to the EGF
receptor
EGFR is phosphorylated
EGFR recruits GBR2 and
forms complex with SOS-1
RAS is activated and initiates
signaling pathway
Phosphotyrosine Response to EGF
Affymetrix Confidential
109
Summary
ƒ
bDNA technology is the basis of FDA-approved viral load assays (HIV and HCV)
and widely adopted in biological research, drug discovery & clinical trials.
ƒ
Proven performance in FDA-commissioned MAQC study demonstrating excellent
correlation with qPCR & microarray platforms.
ƒ
Easy Workflow: QuantiGene® & QuantiGene® Plex work directly from lysates.
ƒ
QuantiGene® Plex measures housekeeping and target genes in the same well for
minimal sample handling & optimal normalization.
ƒ
Numerous applications using QuantiGene® & QuantiGene® Plex including: in situ
RNA-DNA, DNA CNV, Gene Expression HTS – Plex:Plex.
ƒ
Total solution providing sensitive, accurate & precise measurements of genes
from FFPE & whole blood samples enabling retrospective & prospective studies.
ƒ
QuantiGene® ViewRNA enables measurement of single RNA and DNA molecules
in individual cells in a multiplex format.
ƒ
QuantiGene® is linear a assay that easily differentiates 1, 2, and 3 DNA copies.
ƒ
Procarta ® multiplex menu is rapidly growing for protein biomarker profiling &
quantification.
Affymetrix Confidential
110