Validation of a quantitative method for real time PCR kinetics.
Real time RT-PCR is the most sensitive method for quantitation of gene expression levels. The accuracy can be dependent on the mathematical model on which the quantitative methods are based. The generally accepted mathematical model assumes that amplification efficiencies are equal at the exponential phase of the reactions for the same amplicon. However, no methods are available to test the assumptions regarding amplification efficiency before one starts the real time PCR quantitation. Here we further develop and test the validity of a new mathematical model which dynamically fits real time PCR data with good correlation (R(2)=0.9995+/-0.002, n=50). The method is capable of measuring cycle-by-cycle PCR amplification efficiencies and demonstrates that these change dynamically. Validation of the method revealed the intrinsic relationship between the initial amount of gene transcript and kinetic parameters. A new quantitative method is proposed which represents a simple but accurate quantitative method.
Liu
Weihong
W
Department of Physiology, University of Adelaide, Adelaide, SA 5005, Australia.
Saint
David A
DA
D000818:Animals; D002138:Calibration; D003198:Computer Simulation; D018076:DNA, Complementary / Q000737:chemistry; D005456:Fluorescent Dyes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D007700:Kinetics; D008956:Models, Chemical; D008962:Models, Theoretical; D009206:Myocardium / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015221:Potassium Channels / Q000235:genetics; D024683:Potassium Channels, Tandem Pore Domain; D011237:Predictive Value of Tests; D012313:RNA / Q000737:chemistry; D051381:Rats; D015203:Reproducibility of Results
D018076:DNA, Complementary; D005456:Fluorescent Dyes; D015221:Potassium Channels; D024683:Potassium Channels, Tandem Pore Domain; C103742:potassium channel protein TREK-1; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases