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Ffpe pcr array

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  • 1. RT2 Profiler PCR Arrays FFPE & Small Sample Gene Expression Analysis Shankar Sellappan, Ph.D. Global Product Manager 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 the PCR Arrays 2. How PCR Arrays Work with PreAMP 3. PreAMP-PCR Array Performance Data 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. Publish in High Impact Factor Journals 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. Compatible Instrumentation 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 • LightCycler 480 • Mx3000p, Mx3005p, Mx4000p • TP-800 • New Instrument PCR Array Service Core • Set-up protocol available Sample & Assay Technologies
  • 17. Plate Formats & Layouts 96-well, 100-well, 384-well (4x96), 384-well HT Sample & Assay Technologies
  • 18. 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
  • 19. 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 Heat Map 3-D Histogram Register for PCR Array Data Analysis webinar to learn more! Bookmark:http://www.SABiosciences.com/pcr/arrayanalysis.php Sample & Assay Technologies
  • 20. Table of Contents 1. Introduction to the PCR Arrays 2. How PCR Arrays Work With PreAMP Technology 3. PreAMP-PCR Array Performance Data Sample & Assay Technologies
  • 21. How Pathway-Powered PCR Arrays Work Control & Experimental Cells or Tissues 1. Isolate Total RNA 25 ng – 5 ug 2. Reverse Transcription: RT2 First Strand cDNA Synthesis 3. Add RT2 qPCR SYBR Green Master Mix & cDNA Cocktail & Load PCR Array 4. Real-Time PCR (40 cycles) 5. Data Analysis: Determine Fold Changes (∆∆ Ct Method) ∆∆ Sample & Assay Technologies
  • 22. How RT2 PCR Arrays Work: 25 ng – 5 µg RNA RNA (25 ng – 5 ug) 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
  • 23. PCR Arrays: Sensitivity of Detection RT2 Profiler PCR Arrays require Minimum 25 ng Total RNA Positive Call Rate (CT < 35) • 99% with 500 ng RNA • 80% with 25 ng RNA Human Inflammatory Cytokines & Receptors RT2 Profiler PCR Array Sample & Assay Technologies
  • 24. Small or Fixed Samples A Challenge of 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) • FFPE Samples • Even though ample amounts of RNA can be isolated, because of degradation and cross-linking effects, only 1% of recovered RNA is considered suitable for downstream gene expression analysis. Sample & Assay Technologies
  • 25. PCR Arrays with PreAMP – Increased Sensitivity For Small & Fixed Samples See More Genes with Even Less RNA – Pathway-Focused Sample & Assay Technologies
  • 26. How RT2 PCR Arrays Work: 25 ng – 5 µg RNA RNA (25 ng – 5 ug) 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
  • 27. How RT2 PCR Arrays Work Small & FFPE Samples Tandem PCR-based Technology Small Samples (1-100ng RNA) / FFPE Samples (100 ng RNA) 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
  • 28. Essentials of RT2 PreAMP Technology ~40 min + PreAMP Primer Mix and PreAMP Master Mix PreAMP PCR Step (12/8 Cycles) Amplified cDNA ~20 min + Side Reaction Reducer (Exonuclease I) Eliminate Residual PreAMP Primers 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
  • 29. FFPE Samples Gene Expression Challenges RNA Quality • Variable degree of RNA degradation in RNA preparation • Sample handling • Unbuffered fixative solutions • Sub-Optimal Storage & Extraction methods • Impurities Interfere with Enzymatic Activity • Cross-linking with proteins resulting from chemical modification by formaldehyde • Target sequences not available for downstream processes • Prevent enzymes from replicating RNA in a processive manner • RNA is difficult to purify & performs poorly in enzymatic reactions Reduced Effective qRT-PCR templates High Volume (100s) of samples: Biomarkers/ Prognostic models Minimal processing time is essential Sample & Assay Technologies
  • 30. RNeasy FFPE RNA Extraction Kit Proteinase K Digestion: Without standard de-paraffinization, treatment with Proteinase K in a unique buffer system at an elevated temperature reverses cross-links, breaks up and dissolves the tissue sample, and releases the nucleic acids into solution. Phase Extraction: 2 Steps • QiaZol • Guanidinium isothiocyanate • Powerful protein denaturant: Inactivation of RNAses • Acidic Phenol/Chloroform • Partitioning of RNA into aqueous supernatant for separation (low pH) At neutral pH, DNA, not RNA, partitions into the aqueous phase Sample & Assay Technologies
  • 31. FFPE Sample Gene Expression Analysis How it Works Sample & Assay Technologies
  • 32. FFPE Sample Gene Expression Analysis How it Works Sample & Assay Technologies
  • 33. Table of Contents 1. Introduction to the PCR Arrays 2. How PCR Arrays Work with PreAMP 3. PreAMP-PCR Array Performance Data Sample & Assay Technologies
  • 34. RT2 PreAMP Increased Positive Call Rate • Sample: Human Spleen RNA was reverse transcribed to cDNA with (red bars) or without PreAMPlification (purple bars) using RT² PreAMP PCR Master Mix and RT² PreAMP Primer Mix. • PCR Array: Human Cancer PathwayFinder • Unamplified: 55% of genes were virtually undetectable; no genes in the 10-20 Ct range. •FFPE PreAMPlified: Genes with Ct values >30 shift into the visible range of 10-30. Sample & Assay Technologies
  • 35. RT2 PreAMP Detection of “Absent” Genes • Sample: Human Spleen RNA was reverse transcribed to cDNA with (red bars) or without PreAMPlification (purple bars) using RT² PreAMP PCR Master Mix and RT² PreAMP Primer Mix. • PCR Array: Human Cancer PathwayFinder • Unamplified: 14 genes classified as “Absent” (Ct>35) • FFPE PreAMPlified: 14 genes NOW classified as “Present” (Ct<35) Sample & Assay Technologies
  • 36. RT2 PreAMP Unbiased Amplification • Sample: First-strand cDNA was synthesized from the extracted Human Spleen RNA samples, and 1/4 of the RT product was used for pre-amplification with RT² PreAMP PCR Master Mix plus Human Cancer PathwayFinder RT² PreAMP Primer Mix. Unamplified cDNA synthesized from the same spleen and intestine samples were used as the control. FFPE PreAMP amplified and unamplified cDNA samples were then analyzed on the Human Cancer PathwayFinder PCR Array, with the threshold cycle (Ct) obtained. • PCR Array: Human Cancer PathwayFinder PCR Array • Result: Highly Comparable Ct values between FFPE PreAMP and Non-PreAMP samples Sample & Assay Technologies
  • 37. What’s Next – After PCR Arrays • Validate results with more sample and focused set of genes – Custom PCR Arrays • Identify miRNAs that are expressed or regulating your gene of interest – miRNA PCR Arrays • Assess Biological Impact – Cignal™ Reporters • Knockdown Analysis using RNAi – QIAGEN Flexitube/Flexiplate siRNA and SureSilencing shRNA Plasmids • Analyze interaction between DNA & nuclear proteins – ChampionChIP Chromatin Immunoprecipitation PCR Arrays • Quantify secreted proteins in blood plasma sera – ELISArrays Sample & Assay Technologies
  • 38. 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 re-order • 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
  • 39. 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
  • 40. 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
  • 41. Additional Resources E-Learning Center: Recorded Seminars Sample & Assay Technologies