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

Trends Biotechnol. 2019 Jul;37(7):761-774. doi: 10.1016/j.tibtech.2018.12.002. Epub 2019 Jan 14.


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.

Keywords: MIQE guidelines; RT-qPCR; data analysis; data normalization; experimental design; qPCR.

Publication types

  • Review

MeSH terms

  • Costs and Cost Analysis
  • Real-Time Polymerase Chain Reaction / economics
  • Real-Time Polymerase Chain Reaction / methods*
  • Real-Time Polymerase Chain Reaction / standards*
  • Reproducibility of Results
  • Time