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

End-Point Runs (Allelic Discrimination)Amplification PlotThe Amplification plot displays data from real-time runs after signal normalizationand Multicomponent analysis. It contains the tools for setting the baseline andthreshold cycle (C T ) values for the run.What to look for:• Correct baseline and threshold settings – Are the baseline and thresholdvalues set correctly?Identify the components of the amplification curve and set the baseline so thatthe amplification curve growth begins at a cycle number that is greater than thehighest baseline number.IMPORTANT! Do not adjust the default baseline if the amplification curvegrowth begins after cycle 15.Identify the components of the amplification curve and set the threshold so that it is:• Above the background• Below the plateaued and linear regions• Within in the geometric phase of the amplification curve• Irregular amplification – Do all samples appear to have amplified normally? Thethree phases of the amplification curve should be clearly visible in each signal.• Outlying amplification – When the run data is viewed in the C T vs. WellPosition plot, do replicate wells amplify comparably? Wells producing C Tvalues that differ significantly from the average for the associated replicatewells may be considered outliers.If a plate produces non-uniformity between replicates, some samples on the platecould have evaporated. Check the seal of the optical adhesive cover for leaks.End-Point Runs (Allelic Discrimination)TroubleshootingAnalyzed DataRaw DataWhen faced with irregular data, you can use the SDS software to diagnose somechemistry- and instrument-related problems. The following table contains asummary of checks for verifying the integrity of your run data and to help you begintroubleshooting potential problems.The Raw Data Plot displays the raw reporter fluorescence signal (not normalized) forthe selected wells during each cycle of the PCR.What to look for...• Signal tightness and uniformity – Do the raw spectra signals from replicategroups and controls exhibit similar spectral ‘profiles’? If not, the plate orsample block could be contaminated.• Characteristic signal shape – Do the samples peak at the expectedwavelengths? For example, samples containing only FAM dye-labeled TaqManprobes should not produce raw fluorescence in the peak wavelength of the VICdye component. A signal present in wells that do not contain the dye couldindicate that the sample, master mix, or well contains contaminants.• Signal Plateaus – Do any of the signals plateau? Signal plateaus or saturationcan be an indication that a well contains too much template or fluorescent signal.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-13DRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm