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

Chapter 6Analyzing Real-Time DataReal-Time Runs on the 7900HT InstrumentReal-Time RunsQuantitativeRT-PCRAbsoluteQuantificationRelativeQuantificationReal-time is the term used to describe the category of sequence detection runs inwhich the Applied Biosystems 7900HT Fast Real-Time PCR System is used tomeasure the fluorescence of a biological sample during thermal cycling. In contrastto end-point runs, real-time experiments can be used to achieve both qualitative andquantitative measurements. Real-time analysis can be used in combination witheither TaqMan ® or SYBR ® Green 1 double-stranded DNA binding dye reagents for avariety of purposes including quantitative PCR and dissociation curve analysis.Quantitative RT-PCR is a method used to measure small quantities of ribonucleicacid sequences isolated from biological samples. Typical biological samples includecells, tissues, and fluids. During the RT step, reverse transcription of target RNAproduces corresponding complementary DNA (cDNA) sequences. During thesubsequent PCR, the initial concentration of target cDNA is quantified by amplifyingit to a detectable level. The two types of quantitative RT-PCR supported by the7900HT instrument, absolute and relative, are described in detail below.The objective of an absolute quantification experiment is to accurately determine theabsolute quantity of a single nucleic acid target sequence within an unknown sample.The results of an absolute quantification experiment are reported in the same units ofmeasure as the standard used to make them.The objective of relative quantification is to determine the quantity of a singlenucleic acid target sequence within an unknown sample relative to the same sequencewithin a calibrator sample. Relative quantification of target gene expression iscalculated from Applied Biosystems 7900HT Fast Real-Time PCR System datausing one of the following methods:About the Standard Curve MethodThe Standard Curve Method constructs a standard curve similar to that used inabsolute quantification experiments. However, because the goal of relativequantification is to establish a fractional relationship, data produced by relativequantification standards is used differently. For all experimental samples, targetquantity is determined from the standard curve and divided by the target quantity of acalibrator sample. Because the unit from the standard curve drops out, only therelative dilutions of the standard are necessary to determine relative quantities.About the Comparative C T MethodThe SDS software calculates relative levels of gene expression using theComparative C T Method of data analysis. The Comparative C T Method is similar tothe Standard Curve Method except that it uses arithmetic formulas to achieve thesame results for relative quantification. Instead of a standard curve, the ComparativeC T Method calculates relative gene expression using the equation:Relative Quantity = 2 –∆∆C T6-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm