title: Reliable gene expression analysis by reverse transcription-quantitative PCR: reporting and minimizing the uncertainty in data accuracy.
abstract: Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data.
authors.affiliation: Centre for Environmental Sciences, Hasselt University, 3590 Diepenbeek, Belgium tony.remans@uhasselt.be.
authors.forename: Tony
authors.initials: T
authors.lastname: Remans
authors.affiliation: Centre for Environmental Sciences, Hasselt University, 3590 Diepenbeek, Belgium.
authors.forename: Els
authors.initials: E
authors.lastname: Keunen
authors.affiliation: Hasselt University, 3590 Diepenbeek, Belgium.
authors.forename: Geert Jan
authors.initials: GJ
authors.lastname: Bex
authors.affiliation: Centre for Environmental Sciences, Hasselt University, 3590 Diepenbeek, Belgium.
authors.forename: Karen
authors.initials: K
authors.lastname: Smeets
authors.affiliation: Centre for Environmental Sciences, Hasselt University, 3590 Diepenbeek, Belgium.
authors.forename: Jaco
authors.initials: J
authors.lastname: Vangronsveld
authors.affiliation: Centre for Environmental Sciences, Hasselt University, 3590 Diepenbeek, Belgium.
authors.forename: Ann
authors.initials: A
authors.lastname: Cuypers
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D018506:Gene Expression Regulation, Plant; D006801:Humans; D012015:Reference Standards; D015203:Reproducibility of Results; D012107:Research Design / Q000592:standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D035501:Uncertainty
chemical_list: D029681:Arabidopsis Proteins