Pcrarray
1 like403 views
RT2 Profiler PCR Arrays are a real-time PCR technology that allows researchers to study gene expression patterns across biological pathways and processes. The arrays contain pre-designed primer assays for 84 relevant genes as well as controls on a single plate in a 96-well format. The gene content of the arrays is selected based on biological relevance and published associations with relevant pathways. The primer assays on the arrays undergo extensive validation for sensitivity, specificity, reproducibility, and amplification efficiency. The PCR Array system also includes optimized components for RNA isolation, cDNA synthesis, and real-time PCR to provide a complete validated workflow for gene expression analysis from sample to results.
Pcrarray
- 1. RT2 Profiler PCR Arrays An Advanced Real-Time PCR Technology to Empower Your Pathway Analysis Shankar Sellappan, Ph.D. Global Product Manager Gene Expression Analysis Shankar.Sellappan@QIAGEN.com Sample & Assay Technologies
- 2. Welcome to SABiosciences: Who We Are • SABiosciences is now a Company Sample & Assay Technologies
- 3. Central Dogma of Molecular Biology Transcription DNA Genome 10,000 20,000 Genes Translation mRNA Transcriptome Protein Proteome ~ 50,000 proteins Sample & Assay Technologies
- 4. Gene Expression (mRNA) Regulates Biology Biological Processes, such as Apoptosis, Cell Cycle, Inflammation, Signal Transduction, etc., can be identified by specific patterns of gene expression (pathway-focused). Sample & Assay Technologies
- 5. Differential Gene Expression Analysis Q: How to Assess the Expression of Different mRNAs in a Sample and Compare it across multiple samples? A: Sample & Assay Technologies
- 6. PCR Array – Sample Image Sample & Assay Technologies
- 7. Table of Contents 1. Introduction to PCR Arrays 2. How PCR Arrays Work 3. Performance Data from PCR Arrays 4. Research Applications for PCR Arrays Sample & Assay Technologies
- 8. PCR Arrays for ALL Biomedical Researchers Cancer and Apoptosis Cytokines & Inflammation Development & Stem Cells Apoptosis Inflammatory Cytokines Stem Cells Cell Cycle Th17 for Inflammation WNT Signaling / Notch Signaling Human miRNA Array (NEW!) Common Cytokines / Chemokines Terminal Differentiation Markers Breast Cancer & Estrogen Receptor Inflammasomes - NEW! TGFβ / BMP Signaling Tumor Metastasis NF-kB Signaling Pathway Endothelial Cell Biology Epithelial-to-Mesenchymal Transition Th1-Th2-Th3 Osteogenesis Angiogenesis TNF Ligands Growth Factors Cancer Drug Resistance Toll-like Receptors ECM & Adhesion Signal Transduction Toxicology & Drug Metabolism Neuroscience Signal Transduction PathwayFinder Drug Metabolism Neuroscience Ion Channels NFkB Signaling Drug Phase I Enzymes Neurotransmitter Receptors Jak / Stat Signaling Drug Transporters Neurotrophins & Receptors DNA Damage Signaling Oxidative Stress Neurogenesis and Neural Stem Cell Insulin Signaling Stress & Toxicity MAP Kinase Signaling Other Diseases Custom PCR Arrays (H/M/R/Q/D/F) cAMP / Calcium Signaling Atherosclerosis 96-Well, 384-Well Plate p53 Signaling Diabetes 100-Well Disc, 96x96 Chip ** Over 100 Pathway-Powered PCR Arrays Available** Sample & Assay Technologies
- 9. Primer Assays – Laboratory Verification Data Melting/Dissociation Curve • Single Peak Amplification Plot • PCR Efficiency Sample & Assay Technologies
- 10. How Genes on PCR Arrays Are Selected • Biologically relevant gene content • Not simply biochemical pathways or kinase cascades • Published association with the biological or disease pathway gathered from overlapping sources, including: • Multiple Publicly Accessible Databases • Text Mining Relevant Literature • Technically relevant gene content • Use genes that are regulated at the mRNA level • Specific feedback from thought leaders Genes in Signaling Pathways Genes with High Relevance Sample & Assay Technologies
- 11. Relevant Pathway Content on PCR Arrays Sample & Assay Technologies
- 12. Complete PCR Array System – Bench Verified • The entire system has been optimized and bench-verified and bench we guarantee their performance when used together! • RNA isolation kits (verified) • RT2 Profiler PCR Arrays - Bench verified • RT2 1st Strand cDNA synthesis kit – – – – Efficient genomic DNA removal step Built-in reverse transcription control to monitor sample quality A hybrid method of random primers and Oligo dT Fast (20 minutes); High-throughput available • RT2 SYBR Green Master Mixes – Unique buffer system to ensure PCR specificity and efficiency • Free PCR Array Data Analysis software – Web & Excel based Sample & Assay Technologies
- 13. Table of Contents 1. Introduction to PCR Arrays 2. How PCR Arrays Work 3. Performance Data from PCR Arrays 4. Research Applications for PCR Arrays Sample & Assay Technologies
- 14. Principles of qRT-PCR in PCR Arrays Real-Time PCR • Amplify & simultaneously quantify target DNA Reverse Transcription Real-Time PCR • Amplify & simultaneously quantify messenger RNA (mRNA) Ct Values • Threshold Cycle Sample & Assay Technologies
- 15. Anatomy of a PCR Array • 84 Pathway-Specific Genes of Interest • 5 Housekeeping Genes • Genomic DNA Contamination Control • Reverse Transcription Controls (RTC) n=3 • Positive PCR Controls (PPC) n=3 Supported PCR Array Formats • • • • 96-Well Plate 384-Well Plate 100-Well Disc 96x96 Chip Sample & Assay Technologies
- 16. Plate Formats & Layouts 96-well, 100-well, 384-well (4x96), 384-well HT Sample & Assay Technologies
- 17. PCR Arrays Compatible with ALL Types of Samples • Cultured cells • QIAGEN RNeasy Kits with On-Column DNase Treatment • Tissues • QIAZOL followed by RNeasy Kits with On-Column DNase Treatment • Blood samples • QIAGEN PaxGene Blood RNA kit • Small samples • QIAGEN RNeasy Micro Kit & RT2 PreAMP system • Fine needle biopsy (FNB) samples • Laser captured microdissection (LCM) samples • Stem cell clusters or embryoid bodies • Fluorescence-Activated Cell Sorter (FACS) generated cells • FFPE (Formalin-fixed Paraffin-embedded) • QIAGEN RNeasy FFPE Kit & RT2 PreAMP system Sample & Assay Technologies
- 18. How RT2 PCR Arrays Work RNA Isolation RNA Isolation (RNeasy) RNase-Free DNase Treatment Cells & Tissues RNA (25ng- 5ug)* gDNA Elimination Step ~45 minutes First Strand Synthesis 1st strand cDNA + SYBR Green Master Mix 2 hours 15 minutes Real-time PCR Detection of 89 Gene-specific Amplification on RT2 Profiler PCR Array Upload & Analyze Data Sample & Assay Technologies
- 19. The Science Behind RT2 PCR Arrays Sample & Assay Technologies
- 20. PCR Array Compatible RT-PCR Instruments 96-, 100-, 384-, and 96x96 Well Formats • • • 96-Well Blocks: 7000, 7300, 7500, 7700, ViiA 7 FAST 96-Well Blocks: 7500, 7900HT, Step One Plus, ViiA 7 FAST 384-Well Block: 7900HT, ViiA 7 • • iCycler, MyiQ, MyiQ2, iQ5, CFX96, CFX384 Opticon, Opticon 2, Chromo 4 • Mastercycler ep realplex 2/2S/4/4S • BioMark • Rotor-Gene Q, Rotor-Gene 6000 • LightCycler 480 • Mx3000p, Mx3005p, Mx4000p • TP-800 New Instrument Set-up protocol available • No real time PCR machine? PCRArray Service from SABio Sample & Assay Technologies
- 21. RT2 Profiler™ PCR Array Data Analysis • FREE Complete & Easy Data Analysis with Web-Based Software • From Raw Ct Values to Fold Change Results in Multiple Analysis Formats Volcano Plot Scatter Plot Clustergram 3-D Histogram Register for PCR Array Data Analysis webinar to learn more! Sample & Assay Technologies
- 22. Table of Contents 1. Introduction to the PCR Arrays 2. How PCR Arrays Work 3. Performance Data from PCR Arrays – All individual PCR Primer Assays are wet-bench validated for: • Sensitivity – Enhanced Sensitivity with PreAMP Technology • Specificity • Amplification Efficiency • Consistency 4. Research Applications for PCR Arrays Sample & Assay Technologies
- 23. Superb Sensitivity See More Genes with Less RNA • Human Inflammatory Cytokines & Receptors Array used to analyze uninduced cells. Genes represented at very low copy numbers. • 25 ng - 5 µg total RNA per 96-well plate • 99% of genes detectable with > 500 ng • > 80% of genes detectable with 25 ng • Very sensitive assay! Human Inflammatory Cytokines & Receptors PCR Array tested with Universal Reference RNA Conclusion: See gene expression as rare as UN-induced cytokines. Sample & Assay Technologies
- 24. Exquisite Specificity • Assays on Human TGFβ & BMP Signaling Pathway PCR Array used to analyze BMP genes in Universal Reference RNA. • Single peak dissociation curves • Single gel bands of predicted size • High specificity for genes difficult to design specific primers for. Conclusion: Each well in a PCR Array detects a single genespecific product. Sample & Assay Technologies
- 25. High Reproducibility Reproducibility Among Different Users Reproducibility Among Different Instruments Human Drug Metabolism PCR Array in Universal Reference RNA Conclusion: Same raw threshold cycle data can be obtained for the same samples even from different end users at different times or using different instruments. Sample & Assay Technologies
- 26. Uniform Amplification Efficiency Representative set of 500 out of > 4,000 assays used in PCR Arrays Conclusion: Accurate and reliable ∆∆Ct results are guaranteed. Sample & Assay Technologies
- 27. Wide Dynamic Range Human Acetylcholine Receptor α5 Conclusion: Wide dynamic range to accommodate varying levels of initial gene expression. Sample & Assay Technologies
- 28. Replicates: Technical & Biological Technical • Not necessary Save Time & Money • Reproducibility of the PCR Arrays is very high • Results demonstrate that what you are seeing is a result of biology, not technique. • RTC & PPC show technical reproducibility on each plate, and comparable across plates. Biological • Needed to verify the results are a result of biology • At least 3 replicates per sample for statistical analysis • p values Sample & Assay Technologies
- 29. Table of Contents 1. Introduction to the PCR Arrays 2. How PCR Arrays Work 3. Performance Data from PCR Arrays RT2 PreAMP Pathway-Powered PreAMPlification 4. Research Applications for PCR Arrays Sample & Assay Technologies
- 30. Small or Fixed Samples: A Challenge for RT-PCR • Samples containing less than 25 ng • Sources include: • Stem Cells • Fine Needle Biopsies/ Aspirations • Laser Capture Microdissection • Sorted Samples (FACS, selection markers, GFP) • Formalin-fixed Paraffin-embedded samples • Sources include: • Research animals • Clinical samples Sample & Assay Technologies
- 31. PCR Arrays with PreAMP – Increased Sensitivity See More Genes with Even Less RNA – Pathway-Focused Sample & Assay Technologies
- 32. How RT2 PCR Arrays Work Cells & Tissues RNA (25ng- 5ug) gDNA Elimination Step ~45 min First Strand Synthesis 1st strand cDNA + SYBR Green Master Mix 2 hours Real-time PCR Detection of 89 Gene-specific Amplification on RT2 Profiler PCR Array Sample & Assay Technologies
- 33. How RT2 PreAMP Technology Works Tandem PCR-based Technology Cells & Tissues RNA (1ng-100ng); FFPE RNA (100ng) gDNA Elimination Step ~45 min First Strand Synthesis (1/4 of RT product i.e. 5µL) µ 1st strand cDNA ~40 min + PreAMP Primer Mix and PreAMP Master Mix PreAMP PCR Step (Nano:12 Cycles FFPE: 8 Cycles) Amplified cDNA ~20 min Total time:< 2 hr 2 hours + Side Reaction Reducer (Exonuclease I) Eliminate Residual PreAMP Primers Clean Amplified cDNA + SYBR Green Master Mix Real-time PCR Detection of 89 Gene-specific Amplification on RT2 Profiler PCR Array Sample & Assay Technologies
- 34. Essentials of RT2 PreAMP Technology 96-well Pathway-Powered PCR Array • 84 Genes of Interest • 5 Housekeeping Genes • 7 Controls Each PreAMP Primer Mix • 84 Genes of Interest • 1 primer set / gene • 84 primer sets combined RT2 PreAMP: Pathway-Focused Gene Amplification • Faithfully Amplify ONLY those Genes Appearing on Your Array Sample & Assay Technologies
- 35. RT2 PreAMP: Increased Positive Call Rate • Sample: Different amounts of Human universal total RNA were reverse transcribed to cDNA with (red bars) or without PreAMPlification (blue bars) using RT² PreAMP PCR Master Mix and RT² PreAMP cDNA Synthesis Primer Mix. • PCR Array: Human Inflammatory Cytokines and Receptors PCR Array • Unamplified: As little as 25 ng total RNA before PreAMP yields greater than an 80% positive call • Nano PreAMPlified: Increased sensitivity observed with RT2 Profiler PCR Array, allowing analysis of total RNA as little as 1 ng with a positive call rate greater than 90%. Sample & Assay Technologies
- 36. ∆Ct-PreAMP cDNA RT2 PreAMP: Unbiased Amplification Process R2=0.95; Slope=1.01 ∆Ct-Unamplified cDNA • Sample: Human Liver Tumor RNA was reverse transcribed to cDNA with or without PreAMPlification using RT² PreAMP PCR Master Mix and RT² PreAMP cDNA Synthesis Primer Mix. • PCR Array: Human Cancer PathwayFinder • Unamplified cDNA: 500 ng total RNA • Nano PreAMPlified cDNA: 5 ng total RNA • Unamplified cDNA and Nano PreAMPlified cDNA, when reverse transcribed and analyzed on a RT2 Profiler PCR Array, yield similar Ct values for each gene under observation. Sample & Assay Technologies
- 37. Table of Contents 1. Introduction to the PCR Arrays 2. How PCR Arrays Work 3. Performance Data from PCR Arrays 4. Research Applications for PCR Arrays Sample & Assay Technologies
- 38. Publish in High Impact Factor Journals SABiosciences.com/support_publication.php#pcrarray Sample & Assay Technologies
- 39. Application Example 1: Cytokine Profiling Common Cytokine PCR Array: Identified 23 Up-Regulated and 6 Down-Regulated Genes Following PMA and Ionomycin Treatment. Sample & Assay Technologies
- 40. Application Example 2: Tumor Analysis ECM & Cell Adhesion PCR Arrays: Revealed Up- and Down- Regulated Genes in Breast Cancer. Sample & Assay Technologies
- 41. Application Example 3: Toxicity Studies Stress & Toxicity PathwayFinder™ PCR Array: Uncovered Distinct Gene Expression Profiles Associated with Liver Toxicity Caused by 3 PPARγ γ Agonists (Actos, Avandia, and Rezulin). Sample & Assay Technologies
- 42. Create Your Own PCR Arrays: Custom Formats • Choose any gene from the Human, Mouse, Rat, Drosophila, Rhesus macaque, or Dog Genomes 96-well 384-well • Analyze the Genes Most Important To Your Research • Available for any real-time PCR instrument. • Wet-Bench Validated design & performance. • 2 to 3 week turn around time first order • 2 to 3 DAY turn around on reorder • 12 Genes : 8 Samples Same Data Analysis as Catalogued Arrays 24 Genes : 4 Samples 32 Genes : 3 Samples 48 Genes : 2 Samples 96 Genes : 1 Sample Custom 100-discs also available # of samples x 4 Sample & Assay Technologies
- 43. What’s Next - After PCR Arrays • Validate results with more sample and focused set of genes – RT2 Single Primer Assays – RT2 Custom PCR Arrays • Identify Transcription Factors Regulating your Gene – SureFind™ Transcriptome PCR Arrays • MicroRNA studies – RT2 MicroRNA PCR Arrays and assays • Assess Biological Impact – Cignal™ Reporters • Analyze interaction between DNA & nuclear proteins – ChampionChIP Chromatin Immunoprecipitation PCR Arrays • Knockdown Analysis – SureSilencing shRNA Plasmids • Quantify secreted proteins in blood plasma sera – ELISArrays Sample & Assay Technologies
- 44. RT2 PCR Array System SUMMARY • • • • Complete System • RNA isolation kits, PCR Arrays, master mix, first strand kit, and free data analysis software • Systematic controls to monitor sample quality, RT and PCR efficiency • 3 hours from RNA to data analysis Breadth of Pathway Content • Profile the genes and pathways that you really care about • More than 100 pathways for human, mouse, and rat • Customizable by gene, set, or pathway Superb Real-Time PCR Performance • Reproducibility, Sensitivity, Specificity, Reliability Broad Application • Cancer, Immunology, Neuroscience, Stem Cells & much more Sample & Assay Technologies
- 45. Additional Resources E-Learning Center: Recorded Seminars Sample & Assay Technologies
- 46. PCR Arrays EmPower Your Pathway Analysis Experience PCR Array Performance Try them today! Questions? Contact Technical Support 9 AM – 6 PM Eastern M – F Contact: 1-888-503-3187 OR support@SABiosciences.com Shankar Sellappan, Ph.D. (Global Product Manager): Shankar.Sellappan@QIAGEN.com Thousands of scientists have discovered the power of PCR Arrays. Join them on the road to success! Sample & Assay Technologies