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).
One of the potential causes for such a deviating trend is pipetting errors. However, imprecise pipetting would have been detected much earlier based on the coefficients of determination (r2) of the standard curves. Inaccurate pipetting might explain the observed drop in average amplification efficiency over the long run if it occurred in between our pipette calibrations or if the inaccuracy fell within the tolerated range of pipetting inaccuracy. To assess the impact of inaccurate pipetting on amplification efficiency determination using dilution series we performed a mathematical simulation study. This analysis indicates that every percentage of inaccurate pipetting (of the template in a 7-point 10-fold dilution series) (e.g. 9.9 µl instead of 10 µl) results in an efficiency drop of approximately 0.5%. This may seem small, but for a tolerance on pipetting accuracy of 10% this would increase the fraction of failed assays (efficiency below 90%) by 10-fold (from 1.6% to over 16%).
Although the quality and reliability of qPCR, at least when performing relative quantification, does not depend too much on pipetting accuracy (precision remains important though), the assessment of amplification efficiencies from standard curves does require highly accurate pipetting. Indeed, revalidation of our pipets revealed a deviation on one of our pipets requiring adjustment and recalibration, and explaining the observed drop in average amplification efficiency. This event confirms once again the importance of sufficiently frequent pipet calibration to within a small tolerance range for accuracy.