The Ultimate qPCR Experiment: Producing Publication Quality, Reproducible Data the First Time.

Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the methodology is perceived to be relatively simple, there are a number of steps and reagents that require optimization and validation to ensure reproducible data that accurately reflect the biological question(s) being posed. This review article describes and illustrates the critical pitfalls and sources of error in qPCR experiments, along with a rigorous, stepwise process to minimize variability, time, and cost in generating reproducible, publication quality data every time. Finally, an approach to make an informed choice between qPCR and digital PCR technologies is described.

[1]  Raymond R. Panko,et al.  What we know about spreadsheet errors , 1998 .

[2]  F. Speleman,et al.  Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes , 2002, Genome Biology.

[4]  Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA , 2018, Journal of visualized experiments : JoVE.

[5]  Guy Boivin,et al.  Optimization of Droplet Digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations. , 2015, Journal of virological methods.

[6]  W. Vaccaro.  Minimizing liquid delivery risk : Operators as sources of error , 2007 .

[7]  B. Sjögreen,et al.  Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR) , 2017, Biomolecular detection and quantification.

[8]  N. Bissonnette,et al.  Detection limits of several commercial reverse transcriptase enzymes: impact on the low- and high-abundance transcript levels assessed by quantitative RT-PCR , 2007, BMC Molecular Biology.

[9]  Mikael Kubista,et al.  Statistical aspects of quantitative real-time PCR experiment design. , 2010, Methods.

[10]  Jeff Mellen,et al.  High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number , 2011, Analytical chemistry.

[11]  Patrick S. Aranda,et al.  Bleach gel: A simple agarose gel for analyzing RNA quality , 2012, Electrophoresis.

[12]  V. Singer,et al.  RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterization. , 1998, Analytical biochemistry.

[13]  B. H. Miller,et al.  Coordinated Transcription of Key Pathways in the Mouse by the Circadian Clock , 2002, Cell.

[14]  Nicholas C. Wu,et al.  A benchmark study on error-correction by read-pairing and tag-clustering in amplicon-based deep sequencing , 2016, BMC Genomics.

[15]  Tanja Dreo,et al.  Critical points of DNA quantification by real-time PCR--effects of DNA extraction method and sample matrix on quantification of genetically modified organisms. , 2006, BMC biotechnology.

[16]  Sean C Taylor,et al.  The State of RT-Quantitative PCR: Firsthand Observations of Implementation of Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) , 2013, Journal of Molecular Microbiology and Biotechnology.

[17]  Suzanne Kamel-Reid,et al.  Inter-laboratory comparison of chronic myeloid leukemia minimal residual disease monitoring: summary and recommendations. , 2007, The Journal of molecular diagnostics : JMD.

[18]  J. Kleinman,et al.  Spatiotemporal transcriptome of the human brain , 2011, Nature.

[19]  Alaa M Althubaiti,et al.  Information bias in health research: definition, pitfalls, and adjustment methods , 2016, Journal of multidisciplinary healthcare.

[20]  Erik Willems,et al.  The need for transparency and good practices in the qPCR literature , 2013, Nature Methods.

[21]  M. Nikiforova,et al.  Multicenter Comparison of Different Real-Time PCR Assays for Quantitative Detection of Epstein-Barr Virus , 2007, Journal of Clinical Microbiology.

[22]  Julia C. Engelmann,et al.  External calibration with Drosophila whole-cell spike-ins delivers absolute mRNA fold changes from human RNA-Seq and qPCR data. , 2017, BioTechniques.

[23]  D. Dix,et al.  Effects of storage, RNA extraction, genechip type, and donor sex on gene expression profiling of human whole blood. , 2007, Clinical chemistry.

[24]  K M Higgins,et al.  The effect of serial dilution error on calibration inference in immunoassay. , 1998, Biometrics.

[25]  S. Okino,et al.  Evaluation of bias associated with high-multiplex, target-specific pre-amplification , 2015, Biomolecular detection and quantification.

[26]  Tania Nolan,et al.  Talking the talk, but not walking the walk: RT‐qPCR as a paradigm for the lack of reproducibility in molecular research , 2017, European journal of clinical investigation.

[27]  Carle Ab.  Five good reasons: the argument for pipetting technique training. , 2013 .

[28]  Richard Leslie,et al.  GRASP: analysis of genotype-phenotype results from 1390 genome-wide association studies and corresponding open access database , 2014, Bioinform..

[29]  Bruce R McCord,et al.  A Study of PCR Inhibition Mechanisms Using Real Time PCR *,† , 2010, Journal of forensic sciences.

[30]  S A Bustin,et al.  Critical appraisal of quantitative PCR results in colorectal cancer research: Can we rely on published qPCR results? , 2014, Molecular oncology.

[31]  L. Willems,et al.  Interlaboratory Comparison of Six Real-Time PCR Assays for Detection of Bovine Leukemia Virus Proviral DNA , 2018, Journal of Clinical Microbiology.

[32]  Maja Ravnikar,et al.  Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples , 2014, Plant Methods.

[33]  B. Göttgens,et al.  Transcriptional mechanisms of cell fate decisions revealed by single cell expression profiling , 2014, BioEssays : news and reviews in molecular, cellular and developmental biology.

[34]  L. Kaminsky,et al.  Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR. , 1996, BioTechniques.

[35]  T. Godfrey,et al.  Properties of targeted preamplification in DNA and cDNA quantification , 2015, Expert review of molecular diagnostics.

[36]  Alexandra S. Whale,et al.  Fundamentals of multiplexing with digital PCR , 2016, Biomolecular detection and quantification.

[37]  Carole A. Foy,et al.  Considerations for accurate gene expression measurement by reverse transcription quantitative PCR when analysing clinical samples , 2014, Analytical and Bioanalytical Chemistry.

[38]  S. Coulter.  Mitigation of the effect of variability in digital PCR assays through use of duplexed reference assays for normalization. , 2018, BioTechniques.

[39]  John Thomas Bradshaw,et al.  Importance of Integrating a Volume Verification Method for Liquid Handlers: Applications in Learning Performance Behavior , 2007 .

[40]  L. Pelkmans,et al.  Control of Transcript Variability in Single Mammalian Cells , 2015, Cell.

[41]  E. Wolf,et al.  Dynamic changes in messenger RNA profiles of bovine endometrium during the oestrous cycle. , 2008, Reproduction.

[42]  S. Tan,et al.  DNA, RNA, and Protein Extraction: The Past and The Present , 2009, Journal of biomedicine & biotechnology.

[43]  M. Filipecki,et al.  Unintended consequences of plant transformation: A molecular insight , 2010, Journal of Applied Genetics.

[44]  J. Huggett,et al.  Improving the standardization of mRNA measurement by RT-qPCR , 2018, Biomolecular detection and quantification.

[45]  I. Hudecova,et al.  Digital PCR analysis of circulating nucleic acids. , 2015, Clinical biochemistry.

[46]  M. Wiles,et al.  Mouse Estrous Cycle Identification Tool and Images , 2012, PloS one.

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

[48]  Jim F. Huggett,et al.  Reproducibility of biomedical research – The importance of editorial vigilance , 2017, Biomolecular detection and quantification.

[49]  Mikael Kubista,et al.  How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments , 2015, Biomolecular detection and quantification.

[50]  D. Robledo,et al.  Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset , 2014, BMC Genomics.

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

[52]  Tania Nolan,et al.  Variability of the reverse transcription step: practical implications. , 2015, Clinical chemistry.

[53]  Kp Suresh.  An overview of randomization techniques: An unbiased assessment of outcome in clinical research , 2011, Journal of human reproductive sciences.

[54]  Hugo Germain,et al.  Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data , 2017, Scientific Reports.

[55]  K. Gibson,et al.  Measuring and mitigating inhibition during quantitative real time PCR analysis of viral nucleic acid extracts from large-volume environmental water samples. , 2012, Water research.

[56]  Mark F Kavlick,et al.  Development of a universal internal positive control. , 2018, BioTechniques.

[57]  M. Kermekchiev,et al.  Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples , 2009, Nucleic acids research.

[58]  Hye Ryun Woo,et al.  Plant senescence: how plants know when and how to die , 2018, Journal of experimental botany.

[59]  P E Klapper,et al.  Multiplex PCR: Optimization and Application in Diagnostic Virology , 2000, Clinical Microbiology Reviews.

[60]  Lu Wen,et al.  Tracing the temporal-spatial transcriptome landscapes of the human fetal digestive tract using single-cell RNA-sequencing , 2018, Nature Cell Biology.

[61]  C. Foy,et al.  Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples , 2015, Journal of Clinical Microbiology.