Pcr array 2013

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Pcr array 2013

  1. 1. Pathway-powered PCR arrays Advanced Real-Time PCR Technology to Empower Your Pathway Analysis Questions? Comments? or Suggestions? Ask now or contact Technical Support Telephone: 888-503-3187 Email: support@SABiosciences.com Webinar related questions: Qiawebinars@QIAGEN.COM -1- Sample & Assay Technologies
  2. 2. Pathway-focused solution for expression analysis - Complete experimental systems: sample prep through data analysis SABiosciences.com -2- Sample & Assay Technologies
  3. 3. Pathway-focused solution for expression analysis - Complete experimental systems: sample prep through data analysis GeneRead NGS System http://sabiosciences.com/NGS.php Qiagen.com SABiosciences.com -3- Sample & Assay Technologies
  4. 4. Pathway-focused expression analysis - Complete Experimental Systems: Sample Prep through Data Analysis Genes (mRNA) microRNA (miRNA) Cancer Mutation DNA Methylation -4- Sample & Assay Technologies
  5. 5. Gene expression regulates biology All require molecular signaling for action -5- Sample & Assay Technologies
  6. 6. Biological markers define processes Individual participants Cell cycle Angiogenesis Inflammation p53 MDM2 Cyclins CDKs VEGF bFGF ANGPT PDGF IL8 TLR IFNγ TNFβ -6- Sample & Assay Technologies
  7. 7. Complete biological story - Built on pathway / network analysis Angiogenesis Inflammation Pathway Cell cycle -7- Sample & Assay Technologies
  8. 8. Differential gene expression Q: How to assess the expression of different mRNAs in a sample involved in a process and compare it across multiple conditions? A: -8- Sample & Assay Technologies
  9. 9. 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 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 / Drug Transporters Neuroscience Ion Channels NFkB Signaling Drug Phase I Enzymes Neurotransmitter Receptors Jak / Stat Signaling Molecular Toxicology PathwayFinder 384HT Neurotrophins & Receptors DNA Damage Signaling Oxidative Stress Neurogenesis and Neural Stem Cell Insulin Signaling Stress & Toxicity Parkinson’s Disease MAP Kinase Signaling Other Diseases cAMP / Calcium Signaling Atherosclerosis 96-Well, 384-Well Plate p53 Signaling Diabetes 100-Well Disc, 96x96 Chip Custom PCR Arrays (H/M/R/Q/D/F/P/B) Over 140 Pathway- powered PCR arrays available -9- Sample & Assay Technologies
  10. 10. All pathways @ www.SABiosciences.com - 10 - Sample & Assay Technologies
  11. 11. Topics will be covered 1. Introduction to PCR Arrays 2. How PCR Arrays Work 3. Performance of PCR Arrays 4. Applications for PCR Arrays 5. What’s next after PCR Arrays - 11 - Sample & Assay Technologies
  12. 12. PCR Array – plate format Human Inflammatory Cytokines & Receptors RT2 Profiler PCR Array 384-Well 4x96 370 - 12 - Sample & Assay Technologies
  13. 13. Primer assays - Laboratory Verification Data Melting/dissociation curve Single peak Amplification plot PCR efficiency Note: If you are designing your own primers - 13 - STOP Sample & Assay Technologies
  14. 14. qPCR primer assay & custom PCR array - Species Coverage Wet-bench validated assays for all genes in: • Human: H/HS • Mouse: M/MM • Rat: R/RN Custom PCR Array- expandable to the following species Cow (Bos taurus) Chicken (Gallus gallus) Horse (Equus ferus caballus) Zebrafish (Danio rerio) Dog (Canis lupus familiaris) Pig (Sus scrofa) Fruit Fly (Drosophila melanogaster) Rabbit (Oryctolagus cuniculus) Chinese Hamster Ovary (CHO, Cricetulus griseus) cell Rhesus macaque (Macaca mulatta) - 14 - Sample & Assay Technologies
  15. 15. 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 Genes in Signaling Pathways Technically relevant gene content • Use genes that are regulated at the mRNA level • Specific feedback from thought leaders Genes with High Relevance - 15 - Sample & Assay Technologies
  16. 16. How genes on PCR arrays are selected - Published sources http://gncpro.sabiosciences.com/gncpro/gncpro.php - 16 - Sample & Assay Technologies
  17. 17. Relevant pathway content on PCR arrays - 17 - Sample & Assay Technologies
  18. 18. Contents will be covered 1. Introduction to PCR Arrays 2. How PCR Arrays work 3. Performance of PCR Arrays 4. Applications for PCR Arrays 5. What’s next after PCR Arrays - 18 - Sample & Assay Technologies
  19. 19. Principles of qRT-PCR: overview • Real-Time PCR • Amplify and simultaneously quantify target DNA • RT2 PCR (Reverse transcription teal-time PCR) • Amplify and simultaneously quantify mRNA • Ct Values: Threshold Cycle Sample & Assay Technologies
  20. 20. Anatomy of a RT2 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 B2M, HPRT, RPL13A, GAPDH, HGDC - 20 - Sample & Assay Technologies
  21. 21. How RT2 Profiler PCR Arrays work - Experimental workflow Treat cells, for example noncancer or cancer cells control Isolate RNA (Rneasy Kits) RNase-Free DNase Treatment sample Stimulate Cells Isolate RNA Genomic DNA Removal step Reverse Transcription step 5mins + 20 mins Convert Total RNA to cDNA RT-PCR Data Analysis + SYBR Green Master Mix Real-time PCR Detection 2hrs Upload raw data (Ct) and analyze data 15 mins - 21 - Sample & Assay Technologies
  22. 22. Interpretation of Ct values - Fold change calculations: PCR Array results / data 1 Raw Ct Value: Relative expression level of that gene in the sample 2 Data normalization = ∆Ct per Gene = Ct Gene X - Ct HKGs (Avg) 3 Up or down-regulation = ∆∆Ct per Gene = ∆Ct Condition 2 - ∆Ct Condition 1 Fold change results per gene = 2-∆∆Ct Ex. My gene is present 4X more in the treated sample than in control sample. Now not only gene, but GENES in a Pathway (some up, some down) Free complete & easy data analysis with web-based software - 22 - Sample & Assay Technologies
  23. 23. RT2 Profiler PCR Array data analysis - FREE complete & easy analysis with web/excel-based software • Gene content for each pathway is pre-loaded • Custom Arrays: Gene List is all you need • From raw Ct to fold change results in multiple analysis formats Volcano Plot Scatter Plot - 23 - Clustergram 3-D Histogram Sample & Assay Technologies
  24. 24. PCR Arrays compatibility - All sample types • 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) samples QIAGEN RNeasy FFPE Kit & RT2 PreAMP system - 24 - Sample & Assay Technologies
  25. 25. PCR Array compatibility - All 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? PCR Array Service from SABio - 25 - Sample & Assay Technologies
  26. 26. Topics will be covered 1. Introduction to the PCR Arrays 2. How PCR Arrays Work 3. Performance of PCR Arrays – All individual PCR Primer Assays are wet-bench validated for: • • • • • Sensitivity (25 ng – 5 µg) – Enhanced Sensitivity with PreAMP Technology (1 ng) Specificity Reproducibility Amplification Efficiency Wide Dynamic Range 4. Applications for PCR Arrays 5. miRNA PCR Arrays & qBiomarker DNA Mutation PCR Arrays & EpiTect DNA Methylation PCR Arrays - 26 - Sample & Assay Technologies
  27. 27. High sensitivity - 25 ng to 5 ug of RNA * • Human Inflammatory Cytokines & Receptors Array used to analyze uninduced cells. Genes represented at very low copy numbers. • 25 ng - 1 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 *RT2 PreAMP: 1 ng RNA Conclusion: See gene expression as rare as UN-induced cytokines. - 27 - Sample & Assay Technologies
  28. 28. High reproducibility - With different users and different instruments Reproducibility Among Different Instruments Reproducibility Among Different Users 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. - 28 - Sample & Assay Technologies
  29. 29. Uniform high PCR amplification efficiency Why this matters? Cycle Gene 1: Your Design (AE=100%) Gene 1: Friends Design (AE=85%) 0 1 1 1 2 1.85 2 4 3.42 .. 35 3.4x1010 ∆ 2.2 x109 15x Difference Does NOT reflect biology….rather, a technical issue But you would have reported this as a biological finding Conclusion: Accurate and reliable ∆∆Ct results are guaranteed. - 29 - Sample & Assay Technologies
  30. 30. Wide dynamic range Human Acetylcholine Receptor α5 Measure genes that are overexpressed as well as those genes that are underexpressed Conclusion: Wide dynamic range to accommodate varying levels of initial gene expression. - 30 - Sample & Assay Technologies
  31. 31. 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 condition for statistical analysis -> p values - 31 - Sample & Assay Technologies
  32. 32. Topics will be covered 1. Introduction to the PCR Arrays 2. How PCR Arrays Work 3. Performance of PCR Arrays 4. Applications for PCR Arrays 5. What’s next after PCR Arrays - 32 - Sample & Assay Technologies
  33. 33. Application example 1 - Tumor analysis Many questions raised 1. What if knockdown all ↑genes & Induce all ↓ genes? Revert to Normal? 2. Why TIMP3 and MMP3 up together? And why at different rates? 3. Correlations between MMP3 & MMP9? Spend your time Analyzing & Thinking About Your Data! • Revealed up- and down- regulated genes in breast cancer. - 33 - Sample & Assay Technologies
  34. 34. Application example 2 - Drug development / toxicity studies Uncovered distinct gene expression profiles associated with liver toxicity caused by 3 PPARγ agonists (Actos, Avandia, and Rezulin). - 34 - Sample & Assay Technologies
  35. 35. Create your own PCR arrays - Custom formats • Choose any gene from the human, mouse, rat, drosophila, rhesus macaque, dog, or pig 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. • 4 to 6 week turn around time first order (1 - 2 weeks in U.S.) • 1 week turn around on re-order (2 - 3 days in U.S.) • 12 Genes : 8 Samples Same data analysis as cataloged arrays 24 Genes : 4 Samples 32 Genes : 3 Samples 48 Genes : 2 Samples 96 Genes : 1 Sample Custom 100-well discs also available # of samples x 4 - 35 - Sample & Assay Technologies
  36. 36. 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 Responsible Transcription Factors SureFind Transcriptome PCR Arrays MicroRNA studies miScript MicroRNA PCR Arrays and assays Assess Biological Impact CIGNAL™ Reporters Analyze interaction between DNA & nuclear proteins EpiTect Chromatin Immunoprecipitation PCR Arrays Quantify secreted proteins in blood plasma sera ELISArrays Knockdown Analysis SureSilencing shRNA Plasmids & FlexiTube and FlexiPlate siRNA Somatic DNA Mutation Status qBiomarker Somatic DNA Mutation PCR Arrays Copy Number Analysis qBiomarker Copy Number PCR Arrays & Assays New! GeneRead DNAseq Targeted Exon Enrichment Panels for NGS - 36 - Sample & Assay Technologies
  37. 37. www.SABiosciences.com - 37 - Sample & Assay Technologies
  38. 38. www.SABiosciences.com - 38 - Sample & Assay Technologies
  39. 39. Pathway central – 100+ signaling pathway maps QIAGEN website: Geneglobe.com: 500 pathway maps - 39 - Sample & Assay Technologies
  40. 40. New literature search engine - SciGlobe SciGlobe - a more intelligent way to search the literature SciGlobe is an advanced search engine for the biomedical literature. It uses cuttingedge linguistic technology to quickly find and deliver the most relevant search results. SciGlobe search delivers focused content, not just lists of publications. http://sabiosciences.com/sciglobe.php - 40 - Sample & Assay Technologies
  41. 41. PCR array system summary • Complete experimental systems • 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 • 2 hours from RNA to data analysis • Breadth of pathway content • Profile the genes and pathways that you really care about • More than 150 pathways for human, mouse, rat, drosophila, rhesus macaque, dog, pig • Customizable by gene, set, or pathway • Superb real-time PCR performance • Reproducibility, Sensitivity, Specificity, Reliability • Broad application • Cancer, immunology, neuroscience, stem cells & much more - 41 - Sample & Assay Technologies
  42. 42. Thank you for attending Are you ready to try? Please contact us today US and Canada Customers: Contact Technical Support: 9 AM-6 PM M – F EST Call: 1-888-503-3187 Email: support@SABiosciences.com Thousands of scientists have discovered the power of PCR Arrays. Join them on the road to success! - 42 - Sample & Assay Technologies

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