Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is everywhere. Its ubiquity, however, does not mean its practitioners produce consistent and reproducible results that reflect the underlying biology. The exquisite sensitivity of RT-qPCR demands rigorous experimental design; the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were published to help standardize this technique 1, 2. Although a growing number of labs are applying the MIQE criteria to their experiments, many groups still ignore them, thus increasing the potential of publishing artifactual and inaccurate data. In fact, the erroneous conclusions from previous studies have not only misdirected research but have also had poignant societal impact [for example, see 3, 4].
RT-qPCR employs four principal steps:
- Sample extraction from cells or tissue in a rigorous and reproducible manner;
- RNA isolation and purification;
- Reverse transcription to convert the mRNA to cDNA;
- The qPCR reaction to quantify levels of reverse transcribed cDNA in the sample.
Even in the hands of an experienced researcher, any of these steps can be improperly executed resulting in data where the associated interpretations and conclusions do not reflect the biological outcome. This has been shown recently with both human placental and breast cancer studies where even opposite conclusions could be drawn from the data that was entirely due to improper sample validation and reference gene selection 5, 6. The application of a few simple and fast validation tests can assure that the final RT-qPCR data are solid 7.