Instant evaluation of the absolute initial number of cDNA copies from a single real-time PCR curve. Nucleic Acids Res

Ampli®cation of a cDNA product by quantitative PCR (qPCR) is monitored by a ¯uorescent signal proportional to the amount of produced amplicon. The qPCR ampli®cation 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 concentra-tion and the ¯uorescence measured at the plateau, a speci®c ¯uorescence can thus be calculated. As far as probe hydrolysis quantitatively re¯ects amplicon synthesis, this, in turn, makes it possible to convert measured ¯uorescence 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 esti-mated from the ¯uorescence data, with no need to refer to any calibration with known concentrations of target DNA