How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments

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.

[1]  L. Lehmann,et al.  Real-Time PCR in Microbiology: From Diagnosis to Characterization , 2009 .

[2]  Alfred Pingoud,et al.  Real‐Time Polymerase Chain Reaction , 2003, Chembiochem : a European journal of chemical biology.

[3]  Ladislav Pecen,et al.  Quality control for quantitative PCR based on amplification compatibility test. , 2010, Methods.

[4]  D. Jublot,et al.  CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use , 2005, BMC Immunology.

[5]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[6]  Martin Hemberg,et al.  Quantification of mRNA in single cells and modelling of RT-qPCR induced noise , 2008, BMC Molecular Biology.

[7]  Jana L Gevertz,et al.  Mathematical model of real-time PCR kinetics. , 2005, Biotechnology and bioengineering.

[8]  Rasmus Goll,et al.  Evaluation of absolute quantitation by nonlinear regression in probe-based real-time PCR , 2006, BMC Bioinformatics.

[9]  Nadia Lalam,et al.  Estimation of the reaction efficiency in polymerase chain reaction. , 2006, Journal of theoretical biology.

[10]  S. Bustin.  Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. , 2000, Journal of molecular endocrinology.

[11]  C. Daniel.  Calibration Designs for Machines with Carry-Over and Drift , 1975 .

[12]  P. Walsh,et al.  Simultaneous Amplification and Detection of Specific DNA Sequences , 1992, Bio/Technology.

[13]  C. Wittwer,et al.  Continuous fluorescence monitoring of rapid cycle DNA amplification. , 1997, BioTechniques.

[14]  Ales Tichopad,et al.  Validation of kinetics similarity in qPCR , 2011, Nucleic acids research.

[15]  I. Good.  Some Statistical Applications of Poisson's Work , 1986 .

[16]  Michele Guescini,et al.  METHODOLOGY ARTICLE Open Access Methodology article Shape based kinetic outlier detection in real-time , 2022 .

[17]  V. Beneš,et al.  The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. , 2009, Clinical chemistry.

[18]  J. M. Costa-Fernández,et al.  Basic chemometric techniques in atomic spectroscopy , 2009 .

[19]  G. Mortier,et al.  qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data , 2007, Genome Biology.

[20]  Tania Nolan,et al.  SPUD: a quantitative PCR assay for the detection of inhibitors in nucleic acid preparations. , 2006, Analytical biochemistry.

[21]  Kathleen F. Kerr,et al.  The External RNA Controls Consortium: a progress report , 2005, Nature Methods.

[22]  Mariano J. Alvarez,et al.  Model based analysis of real-time PCR data from DNA binding dye protocols , 2007, BMC Bioinformatics.

[23]  Stephen A Bustin,et al.  Reliability of real-time reverse-transcription PCR in clinical diagnostics: gold standard or substandard? , 2009, Expert review of molecular diagnostics.

[24]  Petter Mostad,et al.  Quantitative real-time PCR method for detection of B-lymphocyte monoclonality by comparison of kappa and lambda immunoglobulin light chain expression. , 2003, Clinical chemistry.

[25]  Andreas Krause,et al.  A standard curve based method for relative real time PCR data processing , 2005, BMC Bioinformatics.

[26]  M. Kubista,et al.  Direct Cell Lysis for Single-Cell Gene Expression Profiling , 2013, Front. Oncol..

[27]  Amplification Efficiency of TaqMan ® Gene Expression Assays Application Note TaqMan , 2004 .

[28]  Weihong Tan,et al.  Genomics and proteomics technologies, 22-23 January, 2001, San Jose, USA , 2001 .

[29]  Petter Mostad,et al.  Quantitative Real-Time PCR Method for Detection of B-Lymphocyte Monoclonality by Comparison of κ and λ Immunoglobulin Light Chain Expression , 2003 .

[30]  G Stolovitzky,et al.  Efficiency of DNA replication in the polymerase chain reaction. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[31]  M. Pfaffl,et al.  Standardized determination of real-time PCR efficiency from a single reaction set-up. , 2003, Nucleic acids research.

[32]  S. Bustin,et al.  Quantification strategies in real-time PCR , 2008 .

[33]  J. Peccoud,et al.  Theoretical uncertainty of measurements using quantitative polymerase chain reaction. , 1996, Biophysical journal.

[34]  Franco Magno,et al.  A statistical overview on univariate calibration, inverse regression, and detection limits: Application to gas chromatography/mass spectrometry technique. , 2007, Mass spectrometry reviews.