When Two-Fold Is Not Enough: Quantifying Uncertainty in Low-Copy qPCR.

Accurate interpretation of qPCR data continues to present significant challenges, particularly at low target concentrations where technical variability, stochastic amplification, and efficiency fluctuations confound quantification. The widespread assumption that qPCR outputs are intrinsically reliable, coupled with inconsistent adherence to best-practice guidelines, has exacerbated issues of reproducibility and contributed to misleading conclusions. This may distort pathogen load quantification in diagnostic settings, whilst in gene expression studies, it can lead to overinterpretation of small fold changes. This study presents a systematic, cross-platform evaluation of qPCR performance across a wide dynamic range using defined reaction mixes and technical replicates. We show that calculated copy numbers can closely match expected values over more than three orders of magnitude, but that variability increases markedly at low input concentrations, often exceeding the magnitude of biologically meaningful differences. We conclude that establishing and reporting confidence intervals from the data itself is essential for transparency and for distinguishing reliable quantification from technical noise.