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

Chapter 6Analyzing Real-Time DataOverviewAbout AbsoluteQuantificationEmploying the5´ NucleaseAssayThe Applied Biosystems 7900HT Fast Real-Time PCR System supports real-timeabsolute quantification of nucleic acids using a standard curve method. The objectiveof absolute quantification is to accurately determine the absolute quantity of a singlenucleic acid target sequence within an unknown sample. The results of an absolutequantification experiment are reported in the same unit of measure as the standardused to make them.Absolute quantification on the 7900HT instrument is accomplished through the useof the polymerase chain reaction and the fluorogenic 5´ nuclease assay (seepage D-2). During setup, standards diluted over several orders of magnitude andunknown samples are loaded onto an optical plate containing master mix andTaqMan reagents targeting a specific nucleic acid sequence. The plate is then loadedinto a 7900HT instrument which has been configured to perform a real-time run.During the thermal cycling, the instrument records the emission resulting from thecleavage of TaqMan ® probes in the presence of the target sequence. After the run, theSDS software processes the raw fluorescence data to produce threshold cycle (C T )values for each sample (see page D-6). The software then computes a standard curvefrom the C T values of the diluted standards and extrapolates absolute quantities forthe unknown samples based on their C T values (see below).Note: See Appendix D, “Theory of Operation,” for more information on thefluorogenic 5´ nuclease assay, real-time data collection, or the mathematicaltransformations of sequence detection data.Figure 6-3 illustrates a standard curve generated from a standard TaqMan ® RNase PInstrument Verification Plate. The arrangement of the samples and standards on theplate are shown in “Examples in This Chapter” on page 6-8.Standard Curve Plot2826C T24Unknowns22201.0 E+2 1.0 E+3 1.0 E+4 1.0 E+5Quantity(LogN initial concentration)Figure 6-3 Standard Curve Generated from a TaqMan RNase P InstrumentVerification Plate6-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm