Focus on your Pathway

Transcription

1 PCR ARRAYS RT 2 Profiler TM PCR ARRAYS Apoptosis Cancer Cell Adhesion Cell Signaling Cytokines Disease Inflammation Neuroscience Stem Cells Toxicology Focus on your Pathway

2 PCR ARRAYS: Pathway Analysis with Real-Time PCR Focus on Your Pathway How PCR ARRAYS Work What are PCR Arrays? RT² Profiler PCR Arrays are the most reliable and sensitive gene expression profiling technology for analyzing a focused panel of genes in signal transduction, biological process, or disease related pathways using real-time PCR. How are PCR Arrays Utilized? The RT² Profiler PCR Arrays have been increasingly used in research on cancer, immunology, stem cells, toxicology, biomarker discovery and validation, and phenotypic analysis of cells and transgenic animals. Why PCR Arrays? Simplicity The simplicity of the PCR Arrays makes expression profiling accessible for routine use in every research laboratory with a real-time PCR instrument. 1. Convert Total RNA to cdna. 2. Add cdna to RT 2 qpcr Master Mix & Aliquot Mixture Across PCR Array. 3. Run in Your Real-Time PCR Instrument. 1.E+1 cdna 1 cdna 2 Profile 1 Profile 2 1.E+1 Performance The PCR Arrays have the sensitive, reproducible, specific, and reliable peformance to accurately profile multiple genes simultaneously in 96- or 384-well formats. Relevance PCR Arrays focus on profiling the genes relevant to the pathways or disease states of your interest. Anatomy of a PCR ARRAY Delta Rn 1.E- 1.E-1 1.E-2 1.E Cycle Number 4. Data Analysis. p-value for Fold Change -6 A B A D C D Fold Change Ratio (log 2 ) Delta Rn 1.E- 1.E-1 1.E-2 1.E Cycle Number 1.E+ 1.E-1 CCNE1 1.E-2 CDKN2A FGFR2 TGFB1 1.E-3 ITGB3 1.E-4 MMP9 ITGA2 MCAM 1.E-5 ITGB4 TIMP3 1.E-6 Normal Breast 1.E+ 1.E-1 1.E-2 1.E-3 1.E-4 1.E-5 1.E-6 Breast Tumor Human Inflammatory Cytokines & Receptors RT 2 Profiler PCR Array Complete Systems Available ABCF1 BCL6 C3 C4A C5 CCL1 CCL11 CCL13 CCL15 CCL16 CCL17 CCL18 RT² First Strand Kit: cdna Synthesis CCL19 CCL8 CCL2 CCR1 CCL CCR2 CCL21 CCR3 CCL23 CCR4 CCL24 CCR5 CCL25 CCR6 CCL26 CCR7 CCL3 CCR8 CCL4 CCR9 CCL5 CEBPB CCL7 CRP Eliminates Genomic DNA Insures RT Efficiency & Consistency -Minute Protocol: Quick & Simple CX3CR1 CXCL1 CXCL CXCL11 CXCL12 CXCL13 CXCL14 CXCL2 CXCL3 CXCL5 CXCL6 CXCL9 SYBR Green qpcr Master Mixes ICEBERG IFNA2 IL1F6 IL1F7 IL IL1F8 ILRA ILRB IL1F9 IL1R1 IL13 IL13RA1 IL1RN IL22 IL17C IL5 IL1A IL5RA IL1B IL8 IL1F IL8RA IL1F5 IL8RB Specific Amplification - No Primer Dimers Maximum Amplification Efficiencies Formulated for the Instrument in Your Lab IL9 IL9R LTA LTB LTB4R MIF SCYE1 SPP1 TNF TNFSF5 TOLLIP XCR1 Data Analysis: Convert Raw C t Data to Fold-Change Results B2M HPRT1 RPL13A GAPDH HOUSEKEEPING GENE CONTROLS ACTB HGDC RTC RTC RTC GENOMIC REVERSE TRANSCRIPTION DNA CONTROL CONTROLS PPC PPC POSITIVE PCR CONTROLS PPC Easy-to-Use Web Portal Each well in a PCR Array represents the expression of a gene related to a pathway or disease state. RTC Controls require the use of the RT 2 First Strand Kit. Support@SABiosciences.com

3 PCR ARRAYS: Performance Reproducibility Sensitivity A Input RNA [ng] 5 4 Signal [-d(rfu)/dt] BMP BMP BMP BMP1 8 BMP2 9 BMP A2 A PCR Arrays Yield Highly Reproducible Results Four replicate sets of raw threshold data (1-4) obtained by two different scientists (A & B) at two different times on Human Drug Metabolism RT2 Profiler PCR Arrays Correlation Coefficients are directly compared. The results A4 A2 A3 A1 R2 demonstrate a high degree of corb relation (R >.99) Accuracy BMP2 BMP Uniformly High PCR Amplification Efficiency BMP6 9 BMP Tm [Co] BMP4 Ct from User B Amplification Efficiency [%] 88.6 BMP1 4 4 Single Dissociation Curves 8 B B B Specificity 6 4 PCR Arrays Let You See More Genes with Less RNA Different amounts of universal total RNA were characterized using the Human Inflammatory Cytokines and Receptors PCR Array, and the percentage of detectable genes was calculated for each RNA amount. As little as 25 ng total RNA yields greater than an 8% positive call, even for cytokines expressed at very low levels. B Human Inflammatory Cytokines & Receptors PCR Array B3 4 4 A3 B B1 4 Ct from User A % Positive Calls High Reproducibility Among Different Users Use as little as 25 ng Total RNA BMP5 BMP6 BMP7 9 BMP15 Agarose Gel PCR Arrays Amplify A Single Gene-Specific Product in Every Reaction Universal total RNA was characterized on the TGFβ / BMP Signaling Pathway PCR Array, followed by dissociation (melt) curve analysis. PCR Arrays specifically detect individual genes despite the expression of related gene family members in the same RNA sample Gene PCR Arrays Yield the Most Accurate Results A representative set of assays for 4, genes used in RT2 PCR Arrays have an average amplification efficiency of 99% with a 95% confidence interval from 9-1%. Consistently high amplification efficiencies enable PCR Arrays to accurately analyze multiple genes simultaneously utilizing the ΔΔCt method.

4 PCR ARRAYS: Application Examples Cancer Research: ECM PCR ARRAYS for Breast Cancer Biomarker Discovery Extracellular Matrix & Adhesion Signaling Pathway Breast Tumor TIMP3 MMP3 UP-REGULATED FN1 NO CHANGE ECM & Cell Adhesion CTNND2 SELE MMP9 ITGB3 COL4A2 CNTN1 ADAMTS1 ITGB4 DOWN-REGULATED Normal Breast ECM & Cell Adhesion PCR Arrays Revealed Up- & Down-Regulated Genes in Breast Cancer Total RNA from normal human breast and a human breast tumor were characterized in technical triplicates, and the relative expression levels for each gene in the two samples are plotted against each other in the Scatter Plot. Genes encoding the matrix metallopeptidases (MMP3 & MMP9) and their inhibitors (TIMP3) are up-regulated, while genes encoding integrins (ITGB3 & ITGB4) are down-regulated, by at least three-fold (outside the silver field) in breast tumors relative to normal tissue. Immunology Research: Monitoring Cytokine Induction with PCR ARRAYS Common Cytokine PCR Array Identified 23 Up-Regulated and 6 Down-Regulated Genes Following PMA and Ionomycin Treatment Triplicate total RNA samples from human peripheral blood mononuclear cells (either untreated or stimulated with 5 ng/ml PMA and 1 µg/ml ionomycin for 6 hours) were characterized. Twenty-three cytokine genes are up-regulated (> 5-fold, p <.5) including interleukins, colony stimulating factors, and TNF ligands after 6 hours of stimulation. Six interleukin and TNF ligand genes are down-regulated under the same conditions. STATISTICAL SIGNIFICANCE p value Common Cytokines IL9 IL CSF2 IL1A IFNG IL2 TNF IL1B IL11 IL21 IL22 CSF1 IL3 TNFSF IL13 LTA IL5 PDGFA IL17 TGFB2 TNFSF11 TNFSF13B BMP6 TNFRSPSF11B IFNA5 TNFSF14 IL1F7 BMP fold difference [log 2 ] Common Cytokines Network Toxicity Research: Determining Drug Toxicity with PCR ARRAYS DNA Repair Mechanism Pathway Fold Up-Regulation SrRNA 1ACTB Stress & Toxicity PathwayFinder tm Actos Avandia Rezulin HMOx1 1GADD45A DNAJB4 HSPCA HSPA5 HSPH1 CYP1A1 TNF DDIT3 HSPA1A CSF2 MT2A CRYAB HSPA6 Stress & Toxicity PathwayFinder PCR Array Uncovered Distinct Gene Expression Profiles Associated with Liver Toxicity Caused by 3 PPARγ Agonists RNA from HepG2 cells treated with three different glitazone PPARγ agonists for type 2 diabetes mellitus was characterized, and the results were compared to that of a vehicle (DMSO) control. A withdrawn drug with idiosyncratic liver toxicity (Rezulin) induces very different changes in the expression of stress-related genes than two safer drugs still on the market (Avandia and Actos). USA TEL: FAX: INTERNATIONAL TEL: FAX:

5 What s Your Pathway? Which Real-Time PCR Instrument? Pathway / Topic Focus Alzheimer s Disease Angiogenesis Angiogenic Growth Factors & Angiogenesis Inhibitors Apoptosis Breast Cancer and Estrogen Receptor Signaling Cancer Drug Resistance and Metabolism Cancer mirna Cancer PathwayFinder Cell Cycle Chemokines and Receptors Common Cytokines DNA Damage Signaling Pathway Drug Metabolism Drug Transporters EGF / PDGF Signaling Pathway Extracellular Matrix and Adhesion Molecules Growth Factors Heat Shock Proteins HIV Infection & Host Response Hypoxia Signaling Pathway Inflammatory Cytokines and Receptors Inflammatory Response & Autoimmunity Innate & Adaptive Immune Responses Interferons (IFN) and Receptors JAK / STAT Signaling Pathway MAP Kinase Signaling Pathway Neurogenesis & Neural Stem Cells Neuroscience Ion Channels and Transporters Neurotransmitter Receptors and Regulators Neurotrophins and Receptors NF B Signaling Pathway Nitric Oxide Signaling Pathway Notch Signaling Pathway Osteogenesis Oxidative Stress and Antioxidant Defense p53 Signaling Pathway PI3K / AKT Signaling Pathway Signal Transduction PathwayFinder Stem Cell Stress and Toxicity PathwayFinder TGFβ / BMP Signaling Pathway Th17 for Autoimmunity and Inflammation Th1-Th2-Th3 Toll-Like Receptor Signaling Pathway Tumor Metastasis Tumor Necrosis Factor (TNF) Ligands and Receptors Wnt Signaling Pathway Custom PCR Arrays PCR Array Gene Expression Analysis Service PCR ARRAY Catalog # Human Mouse Rat PAHS-57 PAHS-24 PAHS-72 PAHS-12 PAHS-5 PAHS-4 MAH-2 PAHS-33 PAHS- PAHS-22 PAHS-21 PAHS-29 PAHS-2 PAHS-7 PAHS-4 PAHS-13 PAHS-41 PAHS-76 PAHS-51 PAHS-32 PAHS-11 PAHS-77 PAHS-52 PAHS-64 PAHS-39 PAHS-61 PAHS-44 PAHS-36 PAHS-6 PAHS-31 PAHS-25 PAHS-62 PAHS-59 PAHS-26 PAHS-65 PAHS-27 PAHS-58 PAHS-14 PAHS-45 PAHS-3 PAHS-35 PAHS-73 PAHS-34 PAHS-18 PAHS-28 PAHS-63 PAHS-43 PAMM-57 PAMM-24 PAMM-72 PAMM-12 PAMM-5 PAMM-4 MAM-2 PAMM-33 PAMM- PAMM-22 PAMM-21 PAMM-29 PAMM-2 PAMM-7 PAMM-4 PAMM-13 PAMM-41 PAMM-76 PAMM-51 PAMM-32 PAMM-11 PAMM-77 PAMM-52 PAMM-64 PAMM-39 PAMM-61 PAMM-44 PAMM-36 PAMM-6 PAMM-31 PAMM-25 PAMM-62 PAMM-59 PAMM-26 PAMM-65 PAMM-27 PAMM-58 PAMM-14 PAMM-45 PAMM-3 PAMM-35 PAMM-73 PAMM-34 PAMM-18 PAMM-28 PAMM-63 PAMM-43 PARN-57 PARN-24 PARN-72 PARN-12 PARN-5 PARN-4 - PARN-33 PARN- PARN-22 PARN-21 PARN-29 PARN-2 PARN-7 PARN-4 PARN-13 PARN-41 PARN-76 PARN-51 PARN-32 PARN-11 PARN-77 PARN-52 PARN-64 PARN-39 PARN-61 PARN-44 PARN-36 PARN-6 PARN-31 PARN-25 PARN-62 PARN-59 PARN-26 PARN-65 PARN-27 PARN-58 PARN-14 PARN-45 PARN-3 PARN-35 PARN-73 PARN-34 PARN-18 PARN-28 PARN-63 PARN-43 Supported PCR Instruments Applied Biosystems (ABI) 7, 7, 75, 75 FAST, 77, 79HT, StepOnePlus (96- & 384-well blocks) Bio-Rad icycler, iq5, MyiQ, Chromo4, Opticon, Opticon 2 Stratagene MxP, Mx5P, Mx4 Roche LightCycler 48 (96- & 384-well bocks) Eppendorf Mastercycler ep realplex Complete the PCR ARRAY System Product RT 2 First Strand Kit PCR Arrays Master Mix Pack Size 12 Arrays 2 Arrays (96-well) 12 Arrays (96-well) 24 Arrays (96-well) 4 Arrays (384-well) 2 Arrays 12 Arrays 24 Arrays Price The RT 2 First Strand Kit is required for use with PCR Arrays. PCR Array pack sizes of 12 & 24 offer the best value. $ 199 $ 499 $ 1599 $ 2499 $ 999 RT 2 SYBR Green qpcr Master Mixes are necessary for use with PCR Arrays. The Master Mix is available with ROX, Fluorescein, or SYBR Green only. $ 189 $ 999 $ 1599 Guaranteed Performance Over Pathways Available Focus on your Pathway

6 Angiogenesis Apoptosis Cancer Cell Signaling Cytokines Disease Inflammation Neuroscience Stem Cells Toxicology (Formerly SuperArray Bioscience Corporation) 6951 Executive Way, Frederick, MD 2173 USA The PCR Array Experts + Pathways Available Guaranteed Peformance Phone: Fax: support@sabiosciences.com 9 SABiosciences Corporation All Rights Reserved. RT 2 Profiler PCR Array, RT 2 Real-Time, PathwayFinder, Focus on Your Pathway, Pathways, SABiosciences & the SA logo are trademarks of SABiosciences Corporation. SYBR is a registered trademark of Molecular Probes, Inc. Actos is a registered trademark of Takeda Pharmaceutical Company, Ltd. Avandia is a registered trademark of GlaxoSmithKline. Rezulin is a registered trademark of Parke-Davis Pharmaceuticals, Ltd. ABI and StepOnePlus are registered trademarks of Applera Corporation. Opticon, Chromo4, iq5, icycler and MyiQ are registered trademarks of Bio-Rad Laboratories. MxP, Mx5P, and Mx4 are registered trademarks of Stratagene. LightCycler is a registered trademark of Roche Molecular Systems. Mastercycler is a registered trademark of Eppendorf, Inc.