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

Chapter 8TroubleshootingReal-Time Runs (Quantitative PCR and Dissociation Curves)TroubleshootingAnalyzed DataRaw Data PlotWhen 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 real-time 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-labeledTaqMan ® probes should not produce raw fluorescence in the wavelength of aVIC ® dye component. A signal present in wells that do not contain the dye couldindicate that the sample, master mix, or well contains contaminants.• Characteristic signal growth – As you drag the bar through the PCR cycles, doyou observe growth as expected? Absent growth curves may indicate a pipettingerror (well lacks template).• 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.MulticomponentPlotThe Multicomponent Plot displays a plot of normalized multicomponent data from asingle well of a real-time run. The plot displays the component dye signals thatcontribute to the composite signal for the well.What to look for:• Correct dyes displayed – Does the plot display all dyes as expected? Thepresence of an unexpected dye may be the result of an error in detector setup,such as assigning the wrong reporter or quencher dye.• ROX dye fluorescence level – Does the ROX dye signal fluoresce below thereporter dyes? If not, the lack of reporter fluorescence may be caused by anabsence of probe in the well (a pipetting error).• Background fluorescence – Do all dyes fluoresce above the background? TheBackground signal is a measure of ambient fluorescence. If a dye fails to fluoresceabove the background, it is a strong indication that the well is missing probeslabeled with the dye (well does not contain probe, PCR master mix, or both).• MSE Level – The MSE (mean squared error) is a mathematical representationof how accurately the multicomponented data fits the raw data. The higher theMSE value, the greater the deviation between the multicomponented data andthe raw data.8-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm