from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

PCR was destined to be a quantitative technique. By both theory and practice, a well optimized PCR doubles the amount of product each cycle for many cycles. Early attempts to harness the quantifying power of PCR were limited by dependence on end-point analysis of the products generated, either by removal of an aliquot of the reaction at predetermined cycle numbers (PCR cycle titration) or serial dilution PCR (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Additional attempts were made to measure PCR products in the log phase of the reaction or include a competitive internal control in the reaction. These methods were time-consuming and labor intensive, often using agarose gels to quantify the amount of PCR product and from this determine an initial template concentration (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Real-time PCR greatly simplified quantification. By monitoring fluorescence once each cycle, fluorescence as a surrogate of PCR product amount can be plotted against cycle number. No longer is there a need to physically sample a reaction at multiple cycles or guess when PCR is exponential. By acquiring data at all cycles, exponential data can be selected in retrospect. The exponential region is identified by plotting fluorescence on a log plot and the earliest cycle "significantly above background" chosen to correlate with the initial template amount. Such quantification cycles (Cqs) are usually determined by either a fluorescence threshold or by the maximum second derivative. In either case, these fractional cycle numbers are inversely related to the log of the initial template concentration. Technical aspects of qPCR and performance guidelines have recently been published (For details see: Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).