Real-time, fluorescence-based quantitative PCR: a snapshot of current procedures and preferences

Abstract

Real-time fluorescence-based quantitative PCR has become established as the benchmark technology for the quantification of nucleic acids, offering an immense choice of protocols, chemistries and instruments. However, whilst there are comparatively few technical problems associated with DNA-targeted quantitative PCR, this is not the case for real-time reverse transcription PCR assays, and there is considerable uncertainty regarding biological or clinical relevance of many real-time reverse transcription PCR results. A survey of working practices of nearly 100 delegates carried out prior to the Third qPCR Symposium held in London, UK, April 25–26, 2005, reveals some of the reasons underlying the variability of reported real-time reverse transcription PCR results. Specifically, the survey reveals extensive interlaboratory variation in assay design, validation and analysis that, together with other dubious practices, are the likely cause for the publication of variable results. These results emphasize the urgent need for the establishment of best practice guidelines for this technology, particularly in the context of its mounting adaptation as a high-throughput clinical diagnostic assay.