SuperArray Product Updates 2008

Focus on Your Pathway

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 Best Selling Product:

• Expression analysis tool for a focused panel of genes

• Pre-designed cataloged arrays for over 100 pathways
• Customized panels ranging from 4 to 384 genes
• Compatible with (almost) all real-time PCR instruments

• 96- or 384-well plate of SYBR Green-optimized qPCR assays

• 84 relevant, pathway- or disease-focused genes
• 5 housekeeping genes and three RNA quality controls

• Very simple two-hour protocol that generates:

• Microarray-like multi-gene content & results
• qPCR sensitivity and reproducibility

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 RT2Profiler™ PCR Array Layout
1 2 3 4 5 6 7 8 9 10 11 12
A

G01 G02 G3 G04 G05 G06 G07 G08 G09 G10 G11 G12
G13 G14 G15 G16 G17 G18 G19 G20 G21 G22 G23 G24
B C D E

G25 G26 G27 G28 G29 G30 G31 G32 G33 G34 G35 G36
G37 G38 G39 G40 G41 G42 G43 G44 G45 G46 G47 G48
G49 G50 G51 G52 G53 G54 G55 G56 G57 G58 G59 G60
G61 G62 G63 G64 G65 G66 G67 G68 G69 G70 G71 G72
F G H

G73 G74 G75 G76 G77 G78 G79 G80 G81 G82 G83 G84
HK1 HK2 HK3 HK4 HK5 GDC RTC RTC RTC PPC PPC PPC

• Wells A1-G12 (G01-G84): 84 Pathway-Focused Genes

• Wells H1-H5 (HK1-HK5): 5 Housekeeping Genes
• Wells H6 (GDC): Genomic DNA Control
• Well H7-H9 (RTC): Reverse Transcription Control
• Well H10-H12 (PPC): Positive PCR Control

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 Market Acceptance of PCR Array
• Launched May 2005
• V2.0 November 2006, V3.0 September 2007
• Better Controls, Up-to-Date Sequence Info
• Over 2000 Customers Worldwide
• Current Publication List >200, 30+ for PCR Array
• Most added in last 3 months of 2007
• Expected to grow very rapidly
• See how researchers like you have used
SuperArray technologies in their research [ Go ]
• Conferences / Tradeshows
• More & More Posters with Data Using PCR
Arrays
• More Customers stopping by & pleased with
PCR Array

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Customer Success Story

" The use of the RT2 Profiler Human Th1-Th2-Th3 PCR array has
provided important insight into the effects of cocaine and the
results of the mRNA profiling have correlated well with
additional work on cocaine-mediated changes in cytokine
secretion and T cell function"

Baldwin GC, Professor and Director, UCLA

Presented at the 2008 Meeting of The College on Problems of

Drug Dependence.

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Technology Leaders’ Endorsement
PCR Array Core Facilities Program in US

Baylor College of Medicine

Baylor College of Medicine- Feigin Center

Stanford University
University of Washington

Thomas Jefferson Kimmel Cancer Center

Trudeau Institute
University of Pennsylvania
UCSF
UCLA
The University of Texas Health Science Center-
Houston

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 New PCR Array Pathways
• Brand New Pathways Coming Soon
• Aging and Senescence
• Asthma
• Autoimmunity & Self Tolerance
• Cell Membrane Receptors
• Cell Migration
• Cell Motility
• Circadian Pathways
• Cytoskeleton
• Cytotoxicity
• Gap Junction Proteins
• Genome Stability & DNA Repair
• Glucocorticoid Signaling
• G-Protein Coupled Receptors
• Hedgehog Signaling
• Immunomodulation
• Integrin Signaling
• Ion Channels
• Kinases
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• Lipoprotein Signaling & Cholesterol Metabolism
• Membrane Trafficking
• Metal & Ion Transport
• miRNA Pathway
• Mitochondrial Function
• Neoplastic Tissue
• PKC & Phospholipase Signaling
• Prostate Cancer
• Proteases & Proteinases
• Protein Hydroxylases
• Protein Trafficking
• Proteoglycans
• RNA Interference Pathway
• Signal Transduction Scaffolding Proteins
• Tissue & Cell Regenerative Pathway
• Ubiquitin Degradation
• Vascular Development Pathway
 Useful Resources for Gene Expression Researchers-
Forward to your friends!

Pathway Central
NEW!

- find pathway reviews

and presentation-ready
pathway maps
-overview of SuperArray core technologies

http://www.superarray.com/seminarlist.php http://www.superarray.com/pathwaycentral.php

Real-time PCR PCR Array

RNA interference Analysis Portal
-provide you insights on -useful web-based
these topics based on software for real-time
our expertise PCR data analysis

http://www.superarray.com/support_faq.php http://www.superarray.com/pcr/arrayanalysis.php

Publications Posters

http://www.superarray.com/support_publication.php http://www.superarray.com/support_posters.php

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 New product 2008:

1) RT2 miRNA PCR Array

2) Cignal Reporter Assays
3) ChIP-qPCR Assays

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1) RT2 miRNA qPCR Array & Assay System

Sensitivity:
As little as 0.5 µg total RNA needed
Multi-Sequence Flexibility:
Analyze up to 376 sequences simultaneously
Simplicity:
As easy as a real-time PCR experiment

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 How the miRNA PCR Array Works
Control & Experimental Cells or Tissue

1. Isolate miRNA (MA-01)

2. Reverse Transcription: RT2 miRNA First Strand Kit (MA-03)

3. Setting Up the PCR Array

Instrument-Specific RT2 qPCR SYBR Green Master Mix & cDNA Cocktail
Aliquot across PCR Array

4. Real-Time PCR
(40 cycles, 100-120 min)

5. Data Analysis: Determine Fold Changes (DD Ct Method)

Excel Template & Web Portal NOW Available

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 Small RNA Enrichment
TRIzol Aqueous Phase
Adjust EtOH Conc.

Small RNA Elutes

Large Nucleic Acid Binds
Readjust Eluate
Adjust EtOH Conc.
Small RNA binds
Wash
Elute Small RNA

Appendix Protocol
Purifies Large RNA

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 RT2 miRNA First Strand Kit (MA-03)
miRNA

Poly(A) Polymerase

AAAAAAAA
M1: miRNA RT Primer & ERC Mix TTTTTTTT
M2: 5X miRNA RT Buffer miRNA RT Primer
M3: miRNA RT Enzyme Mix
RNase-free H2O Reverse Transcriptase
Single-Tube 30-min Reaction
TTTTTTTT

Gene-Specific Primer Real-Time PCR

TTTTTTTT
Universal Primer
Universal Reverse Transcription Method:
Same cDNA preparation can be used for ANY gene

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 miRNA PCR Array Performance
Specificity / Discrimination
miRNA ID Sequence
miR-10a UACCCUGUAGAUCCGAAUUUGUG
miR-10b UACCCUGUAGAACCGAAUUUGUG
miRNA-99a AACCCGUAGAUCCGAUCUUGUG
miRNA-100 AACCCGUAGAUCCGAACUUGUG
miR-196a UAGGUAGUUUCAUGUUGUUGGG
miR-196b UAGGUAGUUUCCUGUUGUUGGG

Relative PCR cross-reactivity is calculated from the difference in Ct values

between using target- or isoform-specific primers to detect 105 copies of
specific miRNA target cDNA template.

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 miRNA PCR Array Performance
Specificity / Discrimination

The proprietary primer design of the RT2 miRNA PCR Arrays and Assays
distinguishes miRNA family members with single nucleotide mismatches.

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 Cancer Application Data

Small RNA isolated from human colon tumor and matched adjacent normal
tissue (Biochain) was characterized with on the Human Cancer RT2 miRNA
PCR Array. Many cancer-specifc miRNA are up-regulated in this model.

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 2) Cell-based Cignal Reporter Assays

• Product Line Breadth

• 18 Pathway-Focused Reporter Assays
• 4 Application-Specific 10-Pathway Suites

• Performance
Exceptional Sensitivity, Specificity, Signal-to-Noise
Ratio

• Convenience
Ready-to-use, preformatted transient assays

• Dual Modalities
• Dual-luciferase quantitative endpoint assays
• GFP dynamic live cell assays with single cell
resolution

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 Cignal Applications

• Functional Genomics – What’s the phenotype of my gene?

Does my gene of interest regulate signaling pathway “X”?

• Functional Proteomics – What’s the phenotype of my protein/peptide?

Does my protein, peptide, or growth factor of interest regulate signaling
pathway “X”?

• Drug Screening – What is the mechanism of action of my small molecule

drug candidates?
Do my drug candidates of interest regulate signaling pathway “X”?

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REPORTER DESIGN: Dual-Luciferase Assays

The Transcriptional Regulatory Elements (TREs)

are what establish the
pathway specificity of each reporter

Pathway-targeted transcriptional
regulatory elements (18)

TATA
Reporter Construct TRE box Firefly luciferase

TATA
Normalization Construct CMV box Renilla luciferase

Constitutive transcriptional
regulatory element (1)

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 How Does the Cignal Reporter Assay Work?

3 components:
1) p/w transcription factor-
We have responsive firefly
experimentally luciferase reporter
optimized the TREs
to maximize the 2) Non-inducible firefly
signal-to-noise lucifirase -negative
ratio control

3) Mixture of constitutively
expressing GFP and
firefly lucifierase-
positive control

Constitutively
expressing Renilla
luciferase reporter as
normalizing. construct

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 Cignal Reporter Assays
Pathway Regulatory Element Transcription Factor

C/EBP C/EBP binding site C/EBP

cAMP/PKA CRE CREB
Cell Cycle E2F binding site E2F/DP1
DNA Damage p53 response element p53
Estrogen Receptor ERE Estrogen Receptor
Glucocorticoid Receptor GRE Glucocorticoid Receptor
Hypoxia HIF response element HIF
Type I Interferons ISRE STAT1/STAT2
Interferon Gamma GAS STAT1/STAT1
MAPK/ERK SRE Elk-1/SRF
MAPK/JNK AP1 binding element AP1
c-Myc E-box binding element Myc/Max
NFkB NFkB binding element NFkB
Notch RBP-Jk response element RBP-Jk
PKC/Ca++ NFAT response element NFAT
Retinoic Acid Receptor RARE Retinoic Acid Receptor
TGFβ SMAD response element SMAD2/SMAD3/SMAD4
Wnt TCF/LEF response element TCF/LEF

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 Cignal Cancer Suite

Pathway Regulatory Element Transcription Factor

Cell Cycle E2F binding site E2F/DP1

DNA Damage p53 response element p53
Hypoxia HIF response element HIF
MAPK/ERK SRE Elk-1/SRF
MAPK/JNK AP1 binding element AP1
c-Myc E-box binding element Myc/Max
NFkB NFkB binding element NFkB
Notch RBP-Jk response element RBP-Jk
TGFβ SMAD response element SMAD2/SMAD4
Wnt TCF/LEF response element TCF/LEF

Cignal Cancer Reporter Assay 10-Pathway Suite (luc) CCA-001L

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 Cignal Immune Response Suite

Pathway Regulatory Element Transcription Factor

C/EBP C/EBP binding site C/EBP

cAMP/PKA CRE CREB
Glucocorticoid Receptor GRE Glucocorticoid Receptor
Type I Interferons ISRE STAT1/STAT2
Interferon Gamma GAS STAT1/STAT1
MAPK/ERK SRE Elk-1/SRF
MAPK/JNK AP1 binding element AP1
NFkB NFkB binding element NFkB
PKC/Ca++ NFAT response element NFAT
TGFβ SMAD response element SMAD2/SMAD4

Cignal Immune Response Reporter Assay 10-Pathway Suite (luc) CCA-002L

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 Cignal Growth & Differentiation Suite

Pathway Regulatory Element Transcription Factor

C/EBP C/EBP binding site C/EBP

cAMP/PKA CRE CREB
Cell Cycle E2F binding site E2F/DP1
MAPK/ERK SRE Elk-1/SRF
MAPK/JNK AP1 binding element AP1
c-Myc E-box binding element Myc/Max
NFκB NFκB binding element NFκB
Notch RBP-Jk response element RBP-Jk
TGFβ SMAD response element SMAD2/SMAD4
Wnt TCF/LEF response element TCF/LEF

Cignal Growth & Diff. Reporter Assay 10-Pathway Suite (luc) CCA-003L

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 Cignal Stress & Toxicity Suite

Pathway Regulatory Element Transcription Factor

Cell Cycle E2F binding site E2F/DP1

DNA Damage p53 response element p53
Glucocorticoid Receptor GRE Glucocorticoid Receptor
Hypoxia HIF response element HIF
MAPK/ERK SRE Elk-1/SRF
MAPK/JNK AP1 binding element AP1
c-Myc E-box binding element Myc/Max
NFkB NFkB binding element NFkB
PKC/Ca++ NFAT response element NFAT
TGFβ SMAD response element SMAD2/SMAD4

Cignal Stress & Toxicity Reporter Assay 10-Pathway Suite (luc) CCA-004L

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 3) ChIP-qPCR Array and Assay System

“A ChIP Off The Old (Real-Time PCR) Block”

Speed & Ease:
ChIP Antibody & qPCR Assay already optimized and ready to use
Comprehensive:
Portfolio will eventually represent the 20 most popular ChIP targets – including
transcription factors & histone modifications
High Performance:
Qualified ChIP Antibody & qPCR Assay provide positive specific signals in a well-
defined cell-based experiment when used with our ChIP Prep Kit

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 HOW IT WORKS: Tiling Scheme
TSS (+1) Structural Gene
mRNA
TF BS
gDNA
Distal Promoter Proximal Promoter

1 kb 1 kb 1 kb 1 kb 1 kb
qPCR Assay & Amplicon Amplicon Coverage Range (Tile)

• Many transcription factor binding sites (TF BS) per promoter

• Some known, some yet to be discovered
• Optimal qPCR primer design with stringent criteria needs room
to find best amplicon sequence
• Localizing amplicon near TF BS can be a hindrance
• On average, amplicons located within 500 bp from TFBS
• Most fragmented chromatin about that size anyway

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 ChIP-qPCR Search Portal
New to ChIP? Find your Transcription Factor or Gene.
Currently using ChIP? Find a new assay for your queried target.
http://www.superarray.com/chipqpcr.php

Transcription Factor: Any TF ▼

AND / OR
Gene Symbol:

SUBMIT

ChIP-on-chip / aCGH Validators OR CNV-qPCR Users

Chromosome RefSeq:

Position:

SUBMIT

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 Application Example

• Treat four human cancer cell lines (A549, HepG2, MCF7, PC3)
with anti-cancer agent 5-flourouracil (5-FU)
• Grow & Treat Cells with Biological Replicates
• Monitor p53 signaling pathway gene expression
• Human p53 Signaling RT2 Profiler™ PCR Array
• Verify increases in gene expression with p53 ChIP-qPCR
• Formaldehyde Cross-Link; Lyse Cells & Prepare Chromatin
• Fragment DNA by Sonication to 800-1,000-bp Average Size
• ChIP with Human p53 & RNA Pol II Target-Specific Kits
Plus Non-Immune Serum negative control
• Measure p53 & RNA Pol II enrichment at gene promoters
Plus Negative & Positive Control qPCR Assays

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 Application Example Results

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 Useful Resources for Gene Expression Researchers-
Forward to your friends!

Pathway Central
NEW!

- find pathway reviews

and presentation-ready
pathway maps
-overview of SuperArray core technologies

http://www.superarray.com/seminarlist.php http://www.superarray.com/pathwaycentral.php

Real-time PCR PCR Array

RNA interference Analysis Portal
-provide you insights on -useful web-based
these topics based on software for real-time
our expertise PCR data analysis

http://www.superarray.com/support_faq.php http://www.superarray.com/pcr/arrayanalysis.php

Publications Posters

http://www.superarray.com/support_publication.php http://www.superarray.com/support_posters.php

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