Analysis of qPCR Data: From PCR Efficiency to Absolute Target Quantity

Quantitative Polymerase Chain Reaction (qPCR) is a very sensitive method to determine small amounts of DNA or RNA in experimental, environmental, veterinary, forensic and clinical samples. Despite efforts from the qPCR community to address qPCR variability by recommending standardization of reporting of all steps of a qPCR experiment, most reported qPCR results are still grossly biased. The first part of this paper describes two decades of efforts to remedy this situation by promoting so-called efficiency-corrected qPCR data analysis. Although such analysis leads to less variable qPCR results, the outcome, fluorescence at cycle zero, is difficult to grasp. In the second part, we outline how qPCR analyses can result in Ncopy, the number of copies of the target at the start of the reaction. A newly developed theoretical approach determines Ncopy using the characteristics of the amplification curve and the known concentrations of all reaction components. By including these reaction-mix characteristics in the analysis, this Ncopy is assay-, machine- and laboratory-independent and thus allows direct worldwide comparisons. Moreover, Ncopy provides a very intuitive and easy-to-interpret absolute quantitative result.