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PCR Array Data Analysis Tutorial: qPCR Technology Webinar Series Part 3

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Using actual PCR Array data, this slidedeck presents an easy-to-use and free web-based data analysis tool to calculate fold-differences in gene expression from your raw real-time PCR threshold cycles. Learn how you can look at your results in different formats, including heat map, scatter, volcano, clustergram and multigroup plot.

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PCR Array Data Analysis Tutorial: qPCR Technology Webinar Series Part 3

  1. 1. Sample to Insight RT2 Profiler PCR Array Data Analysis Tutorial Anisha Kharkia Associate Product Manager Biological Research Content Management- GeneGlobe RT2 Profiler PCR Array Data AnalysisTutorial 1
  2. 2. Sample to Insight Welcome to our four-part webinar series on qPCR 2 qPCR technology overview, applications, data analysis and service solutions • Part 1: Introduction to Real-Time PCR (Q-PCR / qPCR/ qrt-PCR) • Part 2: Advanced Real-Time PCR Array Technology – Coding and Noncoding RNA Expression Analysis • Part 3: PCR Array Data Analysis Tutorial • Part 4: Accelerate Your Discovery With QIAGEN Service Solutions for Biomarker Research
  3. 3. Sample to Insight Legal disclaimer QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Servicesor your local distributor.
  4. 4. Sample to Insight Pathway-focused solutions for expression analysis http://www.geneglobe.com http://www.qiagen.com/ Microbial DNA qPCR Arrays RT2 Profiler PCR Array Data AnalysisTutorial Webinar; 4
  5. 5. Sample to Insight GeneGlobe Data Analysis Center – new! Helping you even more with your data analysis • Assign your samples into groups more easily o Sample Manager o Upload a Microsoft Excel file • Display your results more professionally o Improved plots and charts o No need to turn off pop-up blocker • More easily design your next experiment o Upgraded What’s Next • Applied to all array and assay technologies What else you need to know • Upgraded custom PCR arrays and individual qPCR assays • Data upload templates require assay catalog numbers o Be sure to download and use the new Microsoft Excel templates RT2 Profiler PCR Array Data AnalysisTutorial 5
  6. 6. Sample to Insight PCR array anatomy Plate formats – all instruments and contentdepth 4x96 1x384 Rotor-Gene Q RT2 Profiler PCR Array Data AnalysisTutorial 6
  7. 7. Sample to Insight PCR array anatomy Pathway- and gene-specific assays and RT-PCR controls RT2 Profiler PCR Array Data AnalysisTutorial 7 • Pathway-specific genes of interest (GOIs) (84) • Reference (HKG) gene controls (5) • Genomic DNA control (GDC) • Reverse transcription controls (RTCs) (3) • Positive PCR controls (PPCs) (3) Choose your reference genes • Manually • Automatically from reference gene panel or pathway-specificGOIs
  8. 8. Sample to Insight Uploading your data4 Agenda Protocol, baseline and threshold overview1 Organizing your raw data2 Sample experiment3 8 What we have covered5
  9. 9. Sample to Insight How RT2 Profiler PCR Arrays work Brief Protocol Overview Control Sample Synthesize cDNA • Genomic DNA removal step (5 min) • Reverse transcription step (20 min) Load plates • One sample per PCR array • Two minutes with multi-channel pipet Run 40-cycle qPCR program • Standard cycling conditions • All real-time PCR instruments • Two hours Upload and analyze data (FREE) • Raw Ct Values • Fold Change Results o Using ∆∆Ct calculations o Gene Y up- / downregulatedby x-fold RT2 Profiler PCR Array Data AnalysisTutorial 9
  10. 10. Sample to Insight Define baseline and threshold Baseline(for ABI, Stratagene, Bio-Rad and Eppendorf instruments) • Use automated baseline if your instrument has adaptive baseline function or • Manually Set Baseline o Using Linear View o Set range starting at cycle two–three to one–two cycles before earliest amplification o No higher than cycle 15 Threshold value • Use log view • Place in linear phase of amplification curve o Above background signal within lower half to one third of curve • Threshold must be same between runs o Important for PPC and RTC interpretation and for selectingreference genes Roche LC480 instruments • Use second derivative maximum • Export Ct values to a blank spreadsheet (Excel), in a single column per array RT2 Profiler PCR Array Data AnalysisTutorial 10
  11. 11. Sample to Insight Setting baseline Linear view – select“Auto Calculated” RT2 Profiler PCR Array Data AnalysisTutorial 11
  12. 12. Sample to Insight Setting threshold Log view – use the same threshold for all PCR arrays RT2 Profiler PCR Array Data AnalysisTutorial 12
  13. 13. Sample to Insight Uploading your data4 Agenda Protocol, baseline and threshold overview1 Organizing your raw data2 Sample experiment3 13 What we have covered5
  14. 14. Sample to Insight Organize raw Ct values 14 Cataloged PCR arrays Row one • Sample name Row two • Groupings Column A • Well location Column B, C, D, etc. • Raw Ct values for each sample RT2 Profiler PCR Array Data AnalysisTutorial
  15. 15. Sample to Insight Organize raw Ct values 15 Custom RT2 Profiler PCR Arrays RT2 Profiler PCR Array Data AnalysisTutorial Row one • Sample Name Row two • Groupings Column A • Well Location Column B • Gene Symbols Column C • Assay catalog numbers Column D, E, F, etc. • Raw Ct values for each sample
  16. 16. Sample to Insight Organize raw Ct values 16 Individual RT2 qPCR Assays RT2 Profiler PCR Array Data AnalysisTutorial Row one • Sample Name Row two • Groupings Column A • Assay numbering Column B • Gene symbols Column C • Assay catalog numbers Column D, E, F, etc. • Raw Ct values for each sample
  17. 17. Sample to Insight Uploading your data4 Agenda Protocol, baseline and threshold overview1 Organizing your raw data2 Sample experiment3 17 What we have covered5
  18. 18. Sample to Insight Sample experiment – experimental setup Group three biological replicatesinto three groups and one control A B C Control: resting 6 h A B C Group 1: PMA + Ionomycin 6 h A B C Group 2: resting 24 h CA B Group 3: PMA + Ionomycin 24 h RT2 Profiler PCR Array Data AnalysisTutorial 18
  19. 19. Sample to Insight Sample experiment – data analysis overview A Group 3 B CA Group 2 B CA Group 1 B CA Control CB CtGOI – CtHKG ∆Ct 1. Calculate ∆Ct on each array for each GOI (Gene Of Interest) A B C A B C A B C A B C RT2 Profiler PCR Array Data AnalysisTutorial 19
  20. 20. Sample to Insight Sample experiment – data analysis overview A Group 3 B CA Group 2 B CA Group 1 B CA Control CB A B C A B C A B C A B C 1. Calculate ∆Ct on each array for each GOI (Gene Of Interest) 2. Calculate average ∆Ct for each gene within a group ∆Ct + ∆Ct + ∆Ct 3 ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct∆Ct ∆Ct RT2 Profiler PCR Array Data AnalysisTutorial 20 ∆Ct + ∆Ct + ∆Ct 3 ∆Ct + ∆Ct + ∆Ct 3 ∆Ct + ∆Ct + ∆Ct 3
  21. 21. Sample to Insight Sample experiment – data analysis overview A B C A B C A B C A B C 1. Calculate ∆Ct on each array for each GOI (Gene Of Interest) 2. Calculate Average ∆Ct for each gene within a Group 3. Calculate ∆∆Ct for each gene between Groups ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct∆Ct ∆Ct ∆∆Ct = ∆CtGroup 1 – ∆Ctcontrol RT2 Profiler PCR Array Data AnalysisTutorial 21 ∆Ct + ∆Ct + ∆Ct 3 ∆Ct + ∆Ct + ∆Ct 3 ∆Ct + ∆Ct + ∆Ct 3 ∆Ct + ∆Ct + ∆Ct 3 ∆∆Ct = ∆CtGroup 2 – ∆Ctcontrol ∆∆Ct = ∆CtGroup 3 – ∆Ctcontrol
  22. 22. Sample to Insight Sample experiment – data analysis overview A B C A B C A B C A B C ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct ∆Ct∆Ct ∆Ct 1. Calculate ∆CT on each array for each GOI (Gene Of Interest) 2. Calculate Average ∆CT for each gene within a Group 3. Calculate ∆∆CT for each gene between Groups 4. Calculate Fold Change: 2-∆∆Ct RT2 Profiler PCR Array Data AnalysisTutorial 22 ∆∆Ct = ∆CtGroup 1 – ∆Ctcontrol ∆∆Ct = ∆CtGroup 2 – ∆Ctcontrol ∆∆Ct = ∆CtGroup 3 – ∆Ctcontrol ∆Ct + ∆Ct + ∆Ct 3 ∆Ct + ∆Ct + ∆Ct 3 ∆Ct + ∆Ct + ∆Ct 3 ∆Ct + ∆Ct + ∆Ct 3
  23. 23. Sample to Insight Uploading your data4 Agenda Protocol, baseline and threshold overview1 Organizing your raw data2 Sample experiment3 23 What we have covered5
  24. 24. Sample to Insight GeneGlobe Data Analysis Center RT2 Profiler PCRArrayData Analysis live demonstration RT2 Profiler PCR Array Data AnalysisTutorial 24 https://www.qiagen.com/us/geneglobe
  25. 25. Sample to Insight Uploading your data4 Agenda Protocol, baseline and threshold overview1 Organizing your raw data2 Sample experiment3 25 What we have covered5
  26. 26. Sample to Insight Summary 1. Set instrument in absolute quantification or standard curve mode ✓ ABI, Stratagene, Bio-Rad and Eppendorf instruments 2. Set baseline o Set automatically with adaptive baseline or set manually in linear view 3. Set threshold o Lower two thirds of amplification plot in log view. Use same threshold for all PCR arrays o Roche LC480: for items two and three use second derivative maximum 4. Export data into Microsoft Excel. Paste raw Ct values into correct format 5. Upload data to GeneGlobe Data Analysis Center web portal 6. Analyze data o Group technical replicates of same biological condition together o Check QC criteria (PPC, RTC, GDC) o Identify stable reference genes o Review fold change data o Export data and publish results RT2 Profiler PCR Array Data AnalysisTutorial 26 What have we covered today?
  27. 27. Sample to Insight Summary • New Sample Manager o More easily assigns your samples into groups • Improved plots and charts o Display your results more professionally • Upgraded What’s Next o Designs your next experiment • Remember: some templates require assaycatalog numbers • Be sure to download and use the new Microsoft Excel templates RT2 Profiler PCR Array Data AnalysisTutorial 27 New benefits
  28. 28. Sample to Insight Thank You for Attending .Questions? Comments? Suggestions? .Please contact Technical Support . US & Canada . Email: BRCsupport@qiagen.com GeneRead NGS System New productfor microbial research RT2 Profiler PCR Array Data AnalysisTutorial 28 For up-to-date licensing information and product- specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Servicesor your local distributor. .Customers outside North America .Email: SABIO@qiagen.com .Webinar questions .Email: QIAWebinars@qiagen.com

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