Quantification Revisited: What qPCR Efficiency Models Reveal About Data Analysis Integrity.

Amplification efficiency is one of the key parameters in quantitative real-time PCR, as it directly influences the accuracy of both absolute and relative quantification. Amplification efficiency, the fold increase per cycle, is affected by oligonucleotide design, reaction chemistry, sample and template properties, and instrument performance. Consequently, it differs between samples, assays and experimental runs. Although methods for estimating the amplification efficiency have been available for more than two decades, most published qPCR studies continue to assume equal and ideal efficiency across assays. This simplifying assumption introduces efficiency- and expression-dependent error into relative expression and fold-change analyses, contributing to the poor reproducibility observed in many PCR-based studies. This review examines the role of the amplification efficiency in qPCR quantification; the consequences of ignoring, assuming or misapplying efficiency; and the practical implications for data interpretation. The review also considers the development of efficiency estimation models and their implications for contemporary analytical approaches, including emerging data-driven methods for amplification curve analyses.