Standardisation and reporting for nucleic acid quantification

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 levels by RT-qPCR. This affects the integrity of the scientific literature, with consequences for not only basic research, but with potentially major implications for the potential development of molecular diagnostic and prognostic monitoring tools. These issues have been addressed by a set of guidelines that propose a minimum standard for the provision of information for qPCR experiments (‘MIQE’). MIQE aims to systematise current variable qPCR methods into a more consistent format that will encourage detailed auditing of experimental detail, data analysis and reporting principles. General implementation of these guidelines is an important requisite for the maturing of qPCR into a robust, accurate and reliable nucleic acid quantification technology.