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

OverviewAutomatic Outlier RemovalAbout OutlyingReplicate WellsAbout theAutomatic OutlierRemovalRegardingAmplificationBeyondReproducibleLimitsFor any PCR, experimental error may cause some wells to amplify insufficiently ornot at all. These wells typically produce C T values that differ significantly from theaverage for the associated replicate wells. If included in the calculations, the C Tvalues from these wells indicate an incorrect level of relative gene expression byskewing the average for the replicate group.The SDS software offers algorithmic identification and removal of outlying data forstudies consisting of replicate populations of three or more wells. The statisticalmethod used by the software is based on Grubbs outlier removal (also known as theMaximum Normalized Residual Test), which permits the exclusion of a single outlierin a population consisting of as few as three replicates.The software applies the Grubbs tests at different stages in the ∆∆C T calculation,depending on the type of endogenous control used in the study. For non-multiplexstudies, the software removes outliers from replicate groups immediately aftercalculating threshold cycles (C T ). For multiplex studies, the software applies thealgorithm following the ∆C T calculation. Also, if an apparent outlier is within 0.25C T s of the mean for the associated replicate group, the software does not remove it.Note: The outlier removal feature is optional and can be activated from the AnalysisSettings dialog box (see page 6-29). Outliers can also be identified and eliminatedmanually as explained on page 6-49.In addition to the Grubbs tests, the algorithm used by the SDS software featuresadditional rules for dealing with SDS data. Unless the majority of samples in areplicate population are at maximum cycle, the algorithm ignores max cycle wells(wells with C T s equal to or greater than the Maximum cycle value).The experimental reproducibility of C T values for relative quantification assaysdecreases as the starting copy number approaches extremely low levels (less than100 copies). Therefore, PCR targets with low starting copy number and subsequenthigh C T values may have significantly diminished statistical reproducibility. To avoidproblems with reproducibility for low-expression targets, the limits of reproducibilitymust be determined experimentally for each relative quantification assay (all targetsand the endogenous control).Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-19DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm