Why is the PCR amplification efficiency still ignored?

J.M. Ruijter - Sep 12, 2016

Most quantitative PCR (qPCR) results in journals are nowadays presented as Ct, delta Ct (dCt) or delta delta Ct (ddCt) values. Often the correct interpretation of the Y-axis in graphs has to be derived from the numbers given, which are not always in line with the axis title. This title most probably indicates that the axis represents the fold difference of the normalized expression of the target gene between the experimental and control conditions. It is of note that by now the 2 and the minus sign that should be there to turn a delta delta Ct into such a fold-difference have disappeared almost completely from publications. This tendency to avoid formulas has turned quantitative PCR into a field of meaningless numbers. For references to illustrate this, just google delta delta Ct.

Topics: qPCR- PCR efficiency- delta delta Ct

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The impact of pipetting errors on PCR amplification efficiencies

Jan Hellemans - Aug 14, 2014

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 (Bustin et al., Clinical Chemistry, 2009). 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).

Topics: PCR efficiency- pipetting error

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