Accred Qual Assur DOI 10.1007/s00769-011-0769-y REVIEW

Abstract The real-time quantitative polymerase chain reaction (qPCR) is probably the most common molecular technique in use today, having become the method of choice for nucleic acid detection and quantification and underpinning applications ranging from basic research through biotechnology and forensic applications to clinical diagnostics. This key technology relies on fluorescence to detect and quantify nucleic acid amplification products, and its homogeneous assay format has transformed legacy polymerase chain reaction (PCR) from a low-throughput qualitative gel-based technique to a frequently automated, rapid, high-throughput quantitative technology. However, the enormous range of protocols together with frequently inappropriate pre-assay conditions, poor assay design and unsuitable data analysis methodologies are impeding its status as a mature, ‘gold standard ’ technology. This, combined with inconsistent and insufficient reporting procedures, has resulted in the widespread publication of data that can be misleading, in particular when this technology is used to quantify cellular mRNA or miRNA level