Instant evaluation of the absolute initial number of cDNA copies from a single real-time PCR curve.
Amplification of a cDNA product by quantitative PCR (qPCR) is monitored by a fluorescent signal proportional to the amount of produced amplicon. The qPCR amplification curve usually displays an exponential phase followed by a non-exponential phase, ending with a plateau. Contrary to prevalent interpretation, we demonstrate that under standard qPCR conditions, the plateau can be explained by depletion of the probe through Taq polymerase- catalysed hydrolysis. Knowing the probe concentration and the fluorescence measured at the plateau, a specific fluorescence can thus be calculated. As far as probe hydrolysis quantitatively reflects amplicon synthesis, this, in turn, makes it possible to convert measured fluorescence levels in the exponential phase into concentrations of produced amplicon. It follows that the absolute target cDNA concentration initially engaged in the qPCR can be directly estimated from the fluorescence data, with no need to refer to any calibration with known concentrations of target DNA.
Swillens
Stéphane
S
Institut de Recherche Interdisciplinaire, Faculté de Médecine, Université Libre de Bruxelles, CP 602, Belgium. swillens@ulb.ac.be
Goffard
Jean-Christophe
JC
Maréchal
Yoann
Y
de Kerchove d'Exaerde
Alban
A
El Housni
Hakim
H
D000818:Animals; D002138:Calibration; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics*; D005453:Fluorescence; D007334:Insulin-Like Growth Factor I / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D013961:Thyroid Gland / Q000378:metabolism; D013997:Time Factors
D018076:DNA, Complementary; D007334:Insulin-Like Growth Factor I