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

OverviewAlgorithmic Manipulation of Raw DataThe SDS software can analyze raw data immediately after a relative quantificationrun is complete. The term “raw data” refers to the spectral data between 500 nm to660 nm collected by the software during the real-time run. During the analysis, thesoftware automatically applies several mathematical transformations to the raw datato generate a more direct measure of the relationship between the spectral changes inthe unknown samples.MulticomponentingSetting theThreshold andCalling ThresholdCyclesOutlier RemovalGenerating GeneExpressionValuesThe first mathematical transformation involves the conversion of the raw data,expressed in terms of Fluorescent Signal vs. Wavelength, to pure dye componentsusing the extracted pure dye standards. At the same time, the software alsodetermines the contribution of each dye in the raw data using the multicomponentalgorithm.After multicomponenting, the baseline and threshold values must be set for eachdetector in the study. The software allows you to set the baseline and threshold valuesmanually (see page 6-49) or automatically (see page 6-19). The C T values computedusing the baseline and threshold data are the basis for generating gene expressionvalues.For 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 relative quantificationcalculations, these outliers can potentially result in erroneous measurements. Toensure precise relative quantification, replicate groups must be carefully scrutinizedfor outlying wells before the analysis. The software provides options for removingoutliers automatically (see page 6-19) or manually (see page 6-49).The final stage in the analysis is the computation of gene expression values from theC T data. The mathematical process for deriving relative quantification values isbriefly described on page D-8. For detailed information on the derivation of the∆∆C T equation, relative quantification data transformations, or other aspects of5´ nuclease chemistry in relation to relative quantification, Applied Biosystemsrecommends the following references:• For information on performing relative quantification using5´ nuclease chemistry on 7900HT instruments, see:– ABI PRISM 7700 Sequence Detection System User Bulletin #2: RelativeQuantification Of Gene Expression (PN 4303859)– RQ Manager Software User Guide(PN 4351670)– The bibliography of this document for a list of technical papers and research.• For information on the derivation of the ∆∆C T Equation, see:– ABI PRISM 7700 Sequence Detection System User Bulletin #2: RelativeQuantification Of Gene Expression (PN 4303859)Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-17DRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm