Real-Time Runs (Quantitative Pcr And Dissociation Curves) - Applied Biosystems 7900HT User Manual

Chapter 8 Troubleshooting

Real-Time Runs (Quantitative PCR and Dissociation Curves)

Troubleshooting
Analyzed Data
Raw Data Plot
Multicomponent
Plot
8-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
September 1, 2004 11:39 am, CH_Trouble.fm
When faced with irregular data, you can use the SDS software to diagnose some
chemistry- and instrument-related problems. The following table contains a
summary of checks for verifying the integrity of your run data and to help you begin
troubleshooting potential problems.
The Raw Data Plot displays the raw reporter fluorescence signal (not normalized) for
the selected wells during each cycle of the real-time PCR.
What to look for:
• Signal tightness and uniformity – Do the raw spectra signals from replicate
groups and controls exhibit similar spectral 'profiles'? If not, the plate or
sample block could be contaminated.
• Characteristic signal shape – Do the samples peak at the expected
wavelengths? For example, samples containing only FAM
TaqMan ® probes should not produce raw fluorescence in the wavelength of a
VIC ® dye component. A signal present in wells that do not contain the dye could
indicate that the sample, master mix, or well contains contaminants.
• Characteristic signal growth – As you drag the bar through the PCR cycles, do
you observe growth as expected? Absent growth curves may indicate a pipetting
error (well lacks template).
• Signal Plateaus – Do any of the signals plateau? Signal plateaus or saturation
can be an indication that a well contains too much template or fluorescent signal.
The Multicomponent Plot displays a plot of normalized multicomponent data from a
single well of a real-time run. The plot displays the component dye signals that
contribute to the composite signal for the well.
What to look for:
• Correct dyes displayed – Does the plot display all dyes as expected? The
presence 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 the
reporter dyes? If not, the lack of reporter fluorescence may be caused by an
absence of probe in the well (a pipetting error).
• Background fluorescence – Do all dyes fluoresce above the background? The
Background signal is a measure of ambient fluorescence. If a dye fails to fluoresce
above the background, it is a strong indication that the well is missing probes
labeled with the dye (well does not contain probe, PCR master mix, or both).
• MSE Level – The MSE (mean squared error) is a mathematical representation
of how accurately the multicomponented data fits the raw data. The higher the
MSE value, the greater the deviation between the multicomponented data and
the raw data.
DRAFT
™ dye-labeled