Relative quantification
Relative quantificationMichael W. Pfaffl in: Real-time PCR. Published by International University Line (Editor: T. Dorak), p IntroductionReverse transcription (RT) followed by a polymerase chain reaction (PCR)represents the most powerful technology to amplify and detect traceamounts of mRNA (Heid et al., 1996; Lockey, 1998). To quantify these lowabundant expressed genes in any biological matrix the real-time quantita-tive RT-PCR (qRT-PCR) is the method of choice. Real-time qRT-PCR hasadvantages compared with conventionally performed semi-quantitativeend point RT-PCR, because of its high sensitivity, high specificity, goodreproducibility, and wide dynamic quantification range (Higuchi et al.)
constant level of fluorescence or C P acquisition according to the established mathematic algorithm (see Section 3.6). Three general procedures of calculation of the relative quantification ratio are established: 1. The so-called ‘delta C t’ (eqs. 1–2 using ∆C P) or ‘delta-delta C t’ method (eqs. 3–4 using ∆∆C
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