Efficiency-correction is required for accurate qPCR analysis and reporting.

Jan Ruitjer, Rebecca Barnewall, Ian Marsh, Andrew Szentirmay, Jane Quinn, R van Houdt, G Gunst, Maurice van den Hoff

Research output: Contribution to journalArticlepeer-review

Abstract

Background. Quantitative PCR (qPCR) aims to measure the DNA or RNA concentration in diagnostic and biological samples based on the Cq value observed in the amplification curves. Results of qPCR experiments are regularly calculated as if all assays are 100% efficient or reported as just Cq, ΔCq or ΔΔCq values.
Contents. When the reaction shows specific amplification, it should be deemed to be positive, irrespective of the observed Cq. Because the Cq is highly dependent on amplification efficiency, which can vary among targets and samples, accurate calculation of the target quantity and relative gene expression, requires that the actual amplification efficiency is taken into account in the analysis and reports. PCR efficiency is frequently derived from standard curves but this approach is affected by dilution errors and hampered by properties of the standard and the diluent. This affects accurate quantification of clinical and biological samples used in diagnostic applications and collected in challenging conditions. PCR efficiencies determined from individual amplification curves avoid these confounders. To obtain unbiased efficiency-corrected results we recommend absolute quantification with a single undiluted calibrator with known target concentration and efficiency values derived from the amplification curves of the calibrator and the unknown samples. Summary. For meaningful diagnostics or biological interpretation, the reported results of qPCR experiments should be efficiency-corrected. To avoid ambiguity, the
21 MIQE guidelines checklist should be extended to require the methods that were used
22 to 1) determine the PCR efficiency and 2) calculate the reported target quantity and
23 relative gene expression value.
Original languageEnglish
Pages (from-to)1
Number of pages40
JournalClinical Chemistry
Publication statusAccepted/In press - 19 Mar 2021

Grant Number

  • 101985

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