Quantitative reverse transcription PCR (RT-qPCR) is anadaptation of the normal PCR process and is utilised in various applicationssuch as gene expression analysis, pathogen detection, genetic testing anddisease research.
PCR requires the use of double stranded DNA whereas RT-qPCRutilises the use of RNA (either in total form or as purified mRNA). Thus, thisis an adaptation of the original PCR process, which is otherwise limited to theuse of double stranded DNA. Because RT-qPCR can utilise RNA, it requires an extrastep, where the RNA is reverse transcribed by reverse transcriptase to formcDNA. To allow the reverse transcription of RNA, a primer is also required, andthe primer is chosen based on the type of RNA that is used and the length ofthe RNA that will undergo PCR. There are three types of primers that can beutilised: Oligo(dT)s – a stretch of thymine bases which binds to the poly(A)tail of mRNA; Random primers – a stretch of 6-9 bases, which bind at variouspoints along the stretch of RNA; Sequence specific primers – are primers whichtarget specific mRNA sequences. This is in addition to the use of primers, inthe PCR process of both normal PCR and RT-qPCR, thus is a further adaptation ofthe process. The progress of the amplification reaction in RT-qPCR is monitoredusing a fluorescent reporter molecule allowing calculation of the initialtemplate quantity in ‘real time’ as the PCR progresses, as opposed to normalPCR where data collection occurs at the end of the reaction (calculations aremade based on the final fluorescence).
This is a less reliable method used tocollect data, as the efficiency of PCR reactions can decrease during lateramplification cycles as a result of increased accumulation of inhibitors andconsumption of reagents and the data is only collected after 30-40 cycles ofnormal PCR. As a result of calculations happening in ‘real time’, RT-qPCRdoesn’t use gel electrophoresis and thus is an additional adaptation from theinitial process of PCR. Examples of fluorescent reporter molecules include:double stranded DNA binding dye or a dye labelled probe. Further adaptations ofthe process includes the fact that RT-qPCR can be further divided into either aone step process or a two step process: the difference between the two beingthat in the one step process, both the reverse transcription and PCR occur inthe same test tube whereas in the two step process, the two steps occur in twoseparate test tubes. The actual three step process of PCR remains the same inboth RT-qPCR and PCR.