Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

Chapter 6Analyzing Real-Time DataAnalyzing the Study Data8. In the Endogenous Control Detector drop-down list, select the detectorrepresenting the endogenous control for your study.IMPORTANT! The detector used as the endogenous control must be present inall sessions attached to the study.9. Select the option button (Multiplexed or Non-Multiplexed) to indicate the typeof endogenous control used.Note: The Multiplexed or Non-Multiplexed option buttons are active only if thesessions loaded for analysis contain both multiplex and non-multiplex reactions.10. In the RQ Min\Max Confidence drop-down list, select the confidence valueyou want the software to use when generating error bars for the gene expressionplot.11. If desired, select the Export Individual ∆C T option button to instruct the SDSsoftware to display the ∆C T values for individual samples belonging to replicategroups. If the option is not selected, the software displays the average ∆C T forthe replicate group.12. Click to accept the analysis settings and exit.13. Analyze the data as explained in “Analyzing the Data” on page 6-30.Analyzingthe DataDuring the analysis, the software mathematically transforms the raw data to establisha comparative relationship between the spectra changes in the passive reference dyeand those of the reporter dye. Based on that comparison, the software calculates acycle threshold (C T ) for each reaction (endogenous control and unknown).Note: See Appendix D, “Theory of Operation,” for a detailed description of the SDSsoftware mathematical transformation of real-time run data.1. Click (or select Analysis > Analyze).The SDS software analyzes the run data and displays the results in theResults tab.2. Choose from the following:• If you choose to set the baseline and threshold values manually, set themfor each detector on the plate as explained on page 6-46.• If using the AutoC T algorithm to set the baseline and threshold values forthe detectors on the plate, verify that the software has called the settingscorrectly for each detector by following the procedure for manually settingthe baseline and threshold on page 6-46 without adjusting the settings.3. If you choose to remove outliers manually, visualize and eliminate outliers fromthe analyzed run data as explained on page 6-46.6-30 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm