Pcr array data analysis 2013

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Pcr array data analysis 2013

  1. 1. RT2 Profiler™ PCR Array: Web-based data analysis tutorial Questions? Comments? or Suggestions? Ask now or contact Technical Support . . M – F, 9 AM – 6 PM EST. Telephone: 888-503-3187; Email: support@SABiosciences.com . . . International customer: Email: SABIO@QIAGEN.COM . . Any webinar related questions: qiawebinars@qiagen.com . -1- Sample & Assay Technologies
  2. 2. Pathway-Focused Solution for Expression Analysis http://sabiosciences.com/home.php -2- Sample & Assay Technologies
  3. 3. Topics to be Covered Topic I: . Brief Technology and Protocol Overview Topic II (Today): PCR Array Data Analysis • • • • • Define Baseline and Threshold Web Portal Location / Address Upload Raw Ct Data Analyze Data & Controls Export Results -3- Sample & Assay Technologies
  4. 4. Anatomy of a PCR Array 4x96 370 -4- Sample & Assay Technologies
  5. 5. 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 . -5- Sample & Assay Technologies
  6. 6. How RT2 Profiler PCR Arrays Work Isolate Total RNA cDNA Synthesis . Control Hot Sauce – – Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.) Load Plates . 1 Sample per PCR Array 2 minutes with multi-channel pipet Run 40 cycle qPCR Program . Standard cycling conditions for all Real Time PCR Instruments 2 hours Upload and Analyze Data (FREE) . 15 minutes from Raw Ct to Fold Change Data -6- Sample & Assay Technologies
  7. 7. How RT2 Profiler PCR Arrays Work cDNA Synthesis . control Hot Sauce Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.) Load Plates . 1 Sample per PCR Array 2 minutes with multi-channel pipet Run 40 cycle qPCR Program . Standard cycling conditions for all Real Time PCR Instruments 2 hours Upload and Analyze Data (FREE) . 15 minutes from Raw Ct to Fold Change Data -7- Sample & Assay Technologies
  8. 8. How RT2 Profiler PCR Arrays Work cDNA Synthesis . control Hot Sauce Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.) Load Plates . 1 Sample per PCR Array 2 minutes with multi-channel pipet Run 40 cycle qPCR Program . Standard cycling conditions for all Real Time PCR Instruments 2 hours Upload and Analyze Data (FREE) . 15 minutes from Raw Ct to Fold Change Data -8- Sample & Assay Technologies
  9. 9. How RT2 Profiler PCR Arrays Work cDNA Synthesis . control Hot Sauce – – Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.) Load Plates . 1 Sample per PCR Array 2 minutes with multi-channel pipet Run 40 cycle qPCR Program . Standard cycling conditions for all Real Time PCR Instruments 2 hours Upload and Analyze Data (FREE) . 15 minutes from Raw Ct to Fold Change Data -9- Sample & Assay Technologies
  10. 10. How RT2 Profiler PCR Arrays Work cDNA Synthesis . control Hot Sauce – – Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.) Load Plates . 1 Sample per PCR Array 2 minutes with multi-channel pipet Run 40 cycle qPCR Program . Standard cycling conditions for all Real Time PCR Instruments 2 hours Upload and Analyze Data (FREE) . 15 minutes from Raw Ct to Fold Change Data - 10 - Sample & Assay Technologies
  11. 11. How RT2 Profiler PCR Arrays Work cDNA Synthesis . control Hot Sauce – – Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.) Load Plates . 1 Sample per PCR Array 2 minutes with multi-channel pipet Run 40 cycle qPCR Program . Standard cycling conditions for all Real Time PCR Instruments 2 hours Upload and Analyze Data (FREE) . 15 minutes from Raw Ct to Fold Change Data - 11 - Sample & Assay Technologies
  12. 12. How RT2 Profiler PCR Arrays Work control cDNA Synthesis . Hot Sauce – – Genomic DNA Removal Step (5 min.) Reverse Transcription Step (20 min.) Load Plates . 1 Sample per PCR Array 2 minutes with multi-channel pipet Raw C(t) Values . Fold Change Results . Using ∆∆C(t) calculations ABL1 is up/down regulated by x fold - 12 - Sample & Assay Technologies
  13. 13. Topics to be Covered Topic I: . Brief Technology and Protocol Overview Topic II (Today): PCR Array Data Analysis • • • • • Define Baseline and Threshold Web Portal Location / Address Upload Raw Ct Data Analyze Data & Controls Export Results - 13 - Sample & Assay Technologies
  14. 14. Define Baseline and Threshold For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*: Baseline • Use Automated Baseline -(if your instrument has Adaptive Baseline function) OR • Manually Set Baseline -Using Linear View: Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest amplification (with highest cycle being cycle #15) . Threshold Value • Use Log View • Place in 1) Linear phase of amplification curve 2) Above background signal, but within lower half to one third of curve . Export Ct values to blank spread sheet (Excel). . Threshold Must Be Same Between Runs (important for PPC and RTC and selecting house keeping genes) ) . *For Roche LC480: Use Second Derivative Maximum - 14 - Sample & Assay Technologies
  15. 15. Define Baseline and Threshold For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*: Baseline • Use Automated Baseline -(if your instrument has Adaptive Baseline function) OR • Manually Set Baseline -Using Linear View: Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest amplification (with highest cycle being cycle #15) . Threshold Value • Use Log View • Place in 1) Linear phase of amplification curve 2) Above background signal, but within lower half to one third of curve . Export Ct values to blank spread sheet (Excel). . Threshold Must Be Same Between Runs (important for PPC and RTC and selecting house keeping genes) ) . *For Roche LC480: Use Second Derivative Maximum - 15 - Sample & Assay Technologies
  16. 16. Define Baseline and Threshold For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*: Baseline • Use Automated Baseline -(if your instrument has Adaptive Baseline function) OR • Manually Set Baseline -Using Linear View: Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest amplification (with highest cycle being cycle #15) . Threshold Value • Use Log View • Place in 1) Linear phase of amplification curve 2) Above background signal, but within lower half to one third of curve . Export Ct values to blank spread sheet (Excel). . Threshold Must Be Same Between Runs (important for PPC and RTC and selecting house keeping genes) . *For Roche LC480: Use Second Derivative Maximum - 16 - Sample & Assay Technologies
  17. 17. Define Baseline and Threshold For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*: Baseline • Use Automated Baseline -(if your instrument has Adaptive Baseline function) OR • Manually Set Baseline -Using Linear View: Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest amplification (with highest cycle being cycle #15) . Threshold Value • Use Log View • Place in 1) Linear phase of amplification curve 2) Above background signal, but within lower half to one third of curve . Export Ct values to a blank spread sheet (Excel). . Threshold Must Be Same Between Runs (important for PPC and RTC and selecting house keeping genes) . *For Roche LC480: Use Second Derivative Maximum - 17 - Sample & Assay Technologies
  18. 18. Define Baseline and Threshold For ABI, Stratagene, Bio-Rad, and Eppendorf Real-Time PCR Instruments*: Baseline • Use Automated Baseline -(if your instrument has Adaptive Baseline function) OR • Manually Set Baseline -Using Linear View: Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest amplification (with highest cycle being cycle #15) . Threshold Value • Use Log View • Place in 1) Linear phase of amplification curve 2) Above background signal, but within lower half to one third of curve . Export Ct values to blank spread sheet (Excel), in a single column. . Threshold must be same between runs (Important for PPC, RTC and selecting house keeping genes) *For Roche LC480: Use Second Derivative Maximum - 18 - Sample & Assay Technologies
  19. 19. Define Baseline and Threshold *For Roche LC480: Use Second Derivative Maximum Baseline • Use Automated Baseline -(if your instrument has Adaptive Baseline function) OR • Manually Set Baseline -Using Linear View: Set to Cycle #2 or #3 up to 1 or 2 cycle values before earliest amplification (with highest cycle being cycle #15) . Threshold Value • Use Log View • Place in 1) Linear phase of amplification curve 2) Above background signal, but within lower half to one third of curve . Export Ct values to blank spread sheet (Excel), in a single column. . Threshold Must Be Same Between Runs (important for PPC an - 19 - Sample & Assay Technologies
  20. 20. Setting Baseline Select “Auto Calculated” Linear View - 20 - Sample & Assay Technologies
  21. 21. Setting Threshold Log View - 21 - Sample & Assay Technologies
  22. 22. Setting Threshold Use the same threshold for All PCR arrays Threshold Line C(t) - 22 - Sample & Assay Technologies
  23. 23. 2 Ways to “CRUNCH” the Data Excel Based Templates •Free! •Download from http://www.sabiosciences.com/pcrarraydataanalysis.php •Good for 2 Group Comparisons (Control + Experimental) •10 PCR Arrays Web-Based Data Analysis •Free! •Upload Excel spreadsheet at: http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php •Good for 100 Group Comparisons (Control + 99 Experimental) •100 PCR Arrays Total http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php - 23 - Sample & Assay Technologies
  24. 24. Web portal location http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php - 24 - Sample & Assay Technologies
  25. 25. Organize Raw C(t) values Download Excel Template from SABiosciences’ Web Portal…or make your own. Cataloged Array Row 1 Sample Name Column A: Well Location Column B,C,D,E… Raw C(t) Values - 25 - Sample & Assay Technologies
  26. 26. Organe Raw C(t) values Download Excel Template from SABiosciences’ Web Portal…or make your own. Custom Array Row 1 Sample Name Column A: Well Location Column B: Gene Name Column C,D,E… Raw C(t) Values Custom Array format can be adapted for Individual PCR Assays - 26 - Sample & Assay Technologies
  27. 27. Organize Raw C(t) values Download Excel Template from SABiosciences’ Web Portal…or make your own. LEAVE BLANK Individual Assays Row 1 Sample Name Column A: Well Location Column B: Gene Name Well # Column C,D,E… Raw C(t) Values - 27 - Sample & Assay Technologies
  28. 28. Our Experiment – An Example Control A B A Group 3 C B A B Group 2 C B Group 1 C A C Time 0 3 biological replicates will be grouped into (3 groups + control) (resting 6 hr) Time 24 hours (resting) - 28 - Sample & Assay Technologies
  29. 29. Our Experiment - Data Analysis Overview Control A B Group 1 C A B Group 2 C A B Group 3 C A B C 3 biological replicates will be grouped into (3 groups + control) 1 PCR Array for Each Sample (12 PCR Arrays Total) - 29 - Sample & Assay Technologies
  30. 30. Our Experiment - Data Analysis Overview Control Group 1 Group 2 B A C A B C A B A C A B C A B B Group 3 A C C B A B C C ∆C(t) C(t)GOI- C(t)HKG 1. Calculate ∆ C(t) for on each array for each GOI (Gene Of Interest) - 30 - Sample & Assay Technologies
  31. 31. Our Experiment - Data Analysis Overview Control Group 1 Group 2 B A ∆C(t) C A B C A B A C A B C A ∆C(t) ∆C(t) ∆C(t)+∆C(t)+∆C(t) 3 1. 2. ∆C(t) ∆C(t) ∆C(t) ∆C(t) B Group 3 B ∆C(t) ∆C(t)+∆C(t)+∆C(t) 3 ∆C(t)+∆C(t)+∆C(t) 3 A C C ∆C(t) B A B ∆C(t) C C ∆C(t) ∆C(t) ∆C(t)+∆C(t)+∆C(t) 3 Calculate ∆ C(t) for on each array for each GOI (Gene Of Interest) Calculate Average ∆ C(t) for each gene within a Group - 31 - Sample & Assay Technologies
  32. 32. Our Experiment - Data Analysis Overview Control A B ∆C(t) Group 1 C ∆C(t) ∆C(t) A ∆C(t) B ∆C(t) Group 2 A C ∆C(t) ∆C(t) B Group 3 C ∆C(t) ∆C(t) A ∆C(t) B C ∆C(t) ∆C(t) ∆C(t)+∆C(t)+∆C(t) 3 ∆C(t)+∆C(t)+∆C(t) 3 ∆C(t)+∆C(t)+∆C(t) 3 ∆C(t)+∆C(t)+∆C(t) 3 1. 2. 3. Calculate ∆ C(t) for on each array for each GOI (Gene Of Interest) Calculate Average ∆ C(t) for each gene within a Group Calculate ∆∆ C(t) for each gene between Groups - 32 - Sample & Assay Technologies
  33. 33. Our Experiment - Data Analysis Overview Control A B ∆C(t) Group 1 C ∆C(t) ∆C(t) A ∆C(t) B ∆C(t) Group 2 A C ∆C(t) ∆C(t) B Group 3 C ∆C(t) ∆C(t) A ∆C(t) B C ∆C(t) ∆C(t) ∆C(t)+∆C(t)+∆C(t) 3 ∆C(t)+∆C(t)+∆C(t) 3 ∆C(t)+∆C(t)+∆C(t) 3 ∆C(t)+∆C(t)+∆C(t) 3 1. 2. 3. 4. Calculate ∆ C(t) for on each array for each GOI (Gene Of Interest) Calculate Average ∆ C(t) for each gene within a Group Calculate ∆∆ C(t) for each gene between Groups ∆∆Ct) ∆∆ Calculate Fold Change: 2(-∆∆ - 33 - Sample & Assay Technologies
  34. 34. Our Experiment-Data Analysis Overview Control (resting 6 hr) Time 24 hours (resting) http://www.sabiosciences.com/manuals/PCRArrayWhitePaper_App.pdf - 34 - Sample & Assay Technologies
  35. 35. Web portal support tools Quick reference 1.Take a Test Run with a Sample Data Set 2. Play a movie http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php - 35 - Sample & Assay Technologies
  36. 36. Web portal support tools http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php - 36 - Sample & Assay Technologies
  37. 37. Upload Raw Ct Data 1.Choose experiment 2. Select Array by catalog number 3. Select excel file with raw CT data http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php - 37 - Sample & Assay Technologies
  38. 38. Analysis setup: define experimental design Define array group (control or group # or exclude) - 38 - Sample & Assay Technologies
  39. 39. Analysis setup: PCR array reproducibility & sample quality 1. Select each group 2. Rotor Gene? PreAMP? 3. Check PPC 4. Check RTC 5. Check GDC - 39 - Sample & Assay Technologies
  40. 40. Analysis setup: PCR array reproducibility & sample quality 1. Select Each Group 2. Rotor Gene? PreAMP? 3. Check PPC Don’t be panic, contact us 4. Check RTC 5. Check GDC - 40 - Sample & Assay Technologies
  41. 41. Analysis setup: Select normalization gene Select Normalization Method - 41 - Sample & Assay Technologies
  42. 42. Method 1: Classic normalization gene selection Manual selection - 42 - Sample & Assay Technologies
  43. 43. How to select/ remove a normalization gene Select normalization gene(s) - 43 - Sample & Assay Technologies
  44. 44. Method 2: automatic selection from the HKG Panel - 44 - Sample & Assay Technologies
  45. 45. Method 2: Automatic selection from the HKG Panel - 45 - Sample & Assay Technologies
  46. 46. Method 3: Automatic selection from whole plate - 46 - Sample & Assay Technologies
  47. 47. Data Setup: Data overview - 47 - Sample & Assay Technologies
  48. 48. Analysis: Analyze results Average ∆Ct Fold Change - 48 - Fold Regulation Sample & Assay Technologies
  49. 49. Analysis: Analyze results Comments - 49 - Sample & Assay Technologies
  50. 50. Visualization of Data Please turn off your Pop-Up Blocker!! - 50 - Sample & Assay Technologies
  51. 51. Visualization of Data – Heat map Choose Groups Click update - 51 - Sample & Assay Technologies
  52. 52. Visualization of Data – Scatter plot - 52 - Sample & Assay Technologies
  53. 53. Visualization of Data – Volcano plot - 53 - Sample & Assay Technologies
  54. 54. Visualization of Data - Clustergram Clustergram - 54 - Sample & Assay Technologies
  55. 55. Summary 1.Set machine in Absolute Quantification / or Standard Curve Mode 2.Set baseline: (ABI, Stratagene, Bio-Rad and Eppendorf*) A. Adaptive (use auto) 3.Set threshold: A. Lower 2/3rds of amplification plot in log view B. Use same threshold for all PCR Arrays *Roche LC480 use Second Derivative Maximum 4. Export data into excel spreadsheet. Paste raw C(t) values into correct form: (sample row 1, C(t)s according to well location.) 5. Upload data to SABiosciences web portal, or download excel data analysis spreadsheets 6. Analyze Data A. Group Biological/Technical Replicates B. QC criteria (PPC, RTC, GDC) C. Focus on Stable Housekeeping Genes D. Fold Change Data E. Export Data and Publish results - 55 - Sample & Assay Technologies
  56. 56. What’s Next - 56 - Sample & Assay Technologies
  57. 57. Thank you for attending our webinar! Questions? Comments? or Suggestions? Please contact Technical Support . . . . . . . . US & Canada Telephone: 888-503-3187 Email: support@SABiosciences.com International Customers Telephone: 888-503-3187 Email: sabio@Qiagen.com GeneRead NGS System - 57 - Sample & Assay Technologies

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