Bioazelle has always validated quantitative PCR assays by combining specificity analysis (targeted sequencing or microfluidic electrophoresis) with an assessment of the PCR amplification efficiency from the slope of a standard curve or dilution series, in accordance with the MIQE guidelines (
). Having wet-lab validated more than 100,000 assays [see tech note 6262 for materials and methods; assays commercialized as PrimePCR assays by Bio-Rad] with an average efficiency of 99% and more than 98% of the assays with an efficiency of at least 90% [Figure 1] we were quite satisfied with the observed performance. However, recently we started to see more assays failing to meet our quality criterion of acceptable PCR efficiency within the 90-110% range. Nothing that would raise an eyebrow for a handful of assays – some assays are simply not good enough – but worrisome when seeing large numbers deviate. Not only did we observe a drop in efficiency but also a concurrent increase in the y-intercept of the standard curve. Once again this could indicate inferior assay performance, but it was suspicious when observed as a trend across thousands of assays (we have a peak wet lab validation capacity of over 2000 assays per week).