Analysis of mRNA expression by real-time PCR

The last few years have witnessed the transformation of the real-time, fluorescence-based reverse transcription polymerase chain reaction (RT-qPCR) from an experimental technology into a mainstream scientific tool for the detection and quantification of RNA with an enormous range of uses in basic research, molecular medicine and biotechnology. The continuous improvement of reagents and instruments, combined with the trend towards high throughput and miniaturisation, is likely to reinforce that pre-eminence and continue to open up new application areas. Nonetheless, although in principle undoubtedly a straightforward technology, the reliability of RT-qPCR assays depends a series of sequential steps that include careful experimental design, optimisation and validation, which must be implemented pragmatically to obtain meaningful, biologically relevant data.