Critical evaluation of methods used to determine amplification efficiency refutes the exponential character of real-time PCR

This study presents a critical evaluation of amplification efficiency determination, which reveals that potentially large underestimations occur when exponential mathematics is applied to the log-linear region. This discrepancy was found to stem from misinterpreting the origin of the log-linear region, which is derived not from an invariant amplification efficiency, but rather from an exponential loss in amplification rate. In contrast, LRE analysis generated Emax estimates that correlated closely to that derived from a standard curve, despite the fact that standard curve analysis is founded upon exponential mathematics. This paradoxical result implies that the quantitative efficacy of positional-based analysis relies not upon the exponential character of real-time PCR, but instead on the ability to precisely define the relative position of an amplification profile.In addition to presenting insights into the sigmoidal character of the polymerase chain reaction, LRE analysis provides a viable alternative to standard curves for amplification efficiency determination, based on analysis of high-quality fluorescence readings within the central region of SYBR Green I generated amplification profiles.All commercial real-time quantitative PCR platforms currently rely on defining the relative position of amplification profiles. As such, they are reliant on amplification of a serially diluted target to provide an estimate of amplification efficiency, which is essential to accurate and reliable quantification [1,2]. However, a major caveat of this approach is that sample-specific inhibitors can compromise both the reliability and accuracy of an assay. This can be a major concern, particularly for samples derived from sources known to contain PCR inhibitors, in that any loss of amplification efficiency will generate unidentified and potentially large quantitative errors [3,4].Driven by the highly desirable ability to assess amplification kinetics within individual PCR reactions