Stephen Andrew Bustin 1,
1Anglia Ruskin University, United Kingdom
Real-time fluorescence-dependent quantitative PCR, with or without a preceding reverse transcription (RT) step, has long been embraced as a valuable and valid means for quantifying nucleic acids. Its results are widely used to report molecular biomarkers of disease, quantify changes to RNA levels in cells or biopsies and iden- tify the tissue of origin from deposits on forensic samples. Clearly, qPCR has an important and effective use for detecting pathogens, SNPs or other DNA-associated applications. However, this accep- tance for RNA is remarkable, given the numerous reports that have emerged over the last twenty years or so that should cast significant doubts on how reliable RT-qPCR data really are. Whilst RT-qPCR can undoubtedly be used to distinguish fairly large differences or changes in nucleic acid levels, the majority of conclusions are based on results that report small changes that are neither robust nor con- sistent. This raises the far-reaching question of whether it is time to abandon RT-qPCR as a method for quantifying RNAs and replace it with more accurate and less error-prone digital PCR technology.
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