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

Chapter 6Analyzing Real-Time Datac. Do one of the following. Select the:Automatic Baseline option button to have the software calculate a baselinefor the selected detector(s).Manual Baseline option button, and enter Start and End cycle values toapply to the selected detector(s).Manual Baseline option button, and leave the Start and End cycles fieldsempty. (You will need to set the values manually as explained on page 6-46.)4. If necessary, repeat steps 2 through 3 for any additional detectors.IMPORTANT! The threshold and baseline values set in step 3 on page 6-11 aredetector specific, and must be set for each individual detector used on in theplate document.5. Click to accept the analysis settings and exit.6. Analyze the data as explained below.Analyzing theRunAfter you have configured the analysis settings, you can analyze the run data. Duringthe analysis, the software mathematically transforms the raw data to establish acomparative relationship between the spectral 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 (standard and unknown). The software thengenerates a standard curve for the run by plotting the standard samples on a graph ofC T versus initial copy number.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 the Resultstab.2. Choose from the following:• 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 as described on page 6-46,• If you choose to set the baseline and threshold values manually, set themfor each detector on the plate as explained on page 6-46.3. If you choose to remove outliers manually, visualize and eliminate outliers fromthe analyzed run data as explained on page 6-46.6-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm