title: How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments.
abstract: We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1-16 qPCR replicates per concentration and we tested 2-10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3-4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.
authors.affiliation: Institute of Biotechnology, Academy of Science of the Czech Republic, Prague, Czech Republic; TATAA Biocenter, Gothenburg, Sweden.
authors.forename: David
authors.initials: D
authors.lastname: Svec
authors.affiliation: Faculty of Medicine Pilsen, Charles University, Pilsen, Czech Republic.
authors.forename: Ales
authors.initials: A
authors.lastname: Tichopad
authors.affiliation: Institute of Biotechnology, Academy of Science of the Czech Republic, Prague, Czech Republic; TATAA Biocenter, Gothenburg, Sweden.
authors.forename: Vendula
authors.initials: V
authors.lastname: Novosadova
authors.affiliation: Physiology Weihenstephan, TUM - Technische Universität München, Freising, Germany.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Pfaffl
authors.affiliation: Institute of Biotechnology, Academy of Science of the Czech Republic, Prague, Czech Republic; TATAA Biocenter, Gothenburg, Sweden.
authors.forename: Mikael
authors.initials: M
authors.lastname: Kubista
keywords: ANCOVA, analysis of covariance; Amplification efficiency; CLSI, Clinical and Laboratory Standards Institute; Cq, cycle of quantification; Dilution series; E, PCR efficiency; EPA, Environmental protection agency; FDA, food and Drug Administration; GMO, genetically modified organism; IEC, International Electrotechnical Commission; ISO, International Organization for Standardization; MIQE, minimum information for publication of quantitative real-time PCR experiments; NTC, no template control; RIN, RNA Integrity Number; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Real-time quantitative PCR; Standard curve; qPCR; qPCR assay validation