Kinetics Quality Assessment for real-time PCR

Abstract: For proper DNA quantification by real-time PCR, compared samples should have similar PCR efficiencies. This assumption might not be always correct, as suggested by hundreds of publications dealing with PCR inhibition. The current methods to ensure proper quantification, normalization with reference genes and Internal Amplification Control (IAC), might not address the problem well. Reference and target genes might be inhibited to different levels with different quantitative effect of the inhibitor. Internal amplification controls are difficult to design and produce, and might severely affect the accuracy and sensitivity of quantification up to 10 folds. Kinetics Quality Assessment (Kinetics QA) is a set of statistical tools to verify the requirement of efficiency similarity. In this work we used Kinetics QA to show that dissimilar efficiencies in compared samples are associated with aberrant quantities. Therefore, we suggest using Kinetics QA to assess real-time PCR quality. Kinetics QA tests do not require additional bench work nor reagents, and they do not affect assay accuracy and sensitivity. However, Kinetics QA tools can be used only at the detectable phase of the reaction and therefore we recommend their use to assign technical reason for aberrant results and not automatically exclude any suspected sample.