Quantitative step, where the RNA is reverse transcribed by

Quantitativereverse transcription PCR (RT-qPCR) is an alteration of the normal PCR processand is used in many applications such as gene expression analysis, pathogendetection, genetic testing and disease research. (ThermoFisher 2017) PCR uses double stranded DNA whereas RT-qPCRutilises the use of RNA (either as pre-mRNA or as purified mRNA). Hence, thisis an adaptation of the original PCR process, which is otherwise restricted to theuse of double stranded DNA. As RT-qPCR utilises RNA, it entails an additionalstep, where the RNA is reverse transcribed by reverse transcriptase to formcDNA. A primer is needed to allow the reverse transcription of RNA and theprimer is selected using the type of RNA that is used and the length of the RNAthat will endure PCR.

There are three kinds of primers that can be used:Oligo(dT)s – a stretch of thymine bases which binds to the poly(A) tail ofmRNA; Random primers – a stretch of 6-9 bases, which bind at various pointsalong the stretch of RNA; Sequence specific primers – are primers which targetspecific mRNA sequences. This is in supplement to the use of primers, in thePCR process of both normal PCR and RT-qPCR, hence it is a further adaptation ofthe process. The progress of the amplification reaction in RT-qPCR is observedusing a fluorescent reporter molecule allowing calculation of the initialtemplate quantity in ‘real time’ as the PCR progresses, as opposed to normalPCR where data collection happens at the end of the reaction (calculations aremade based on the final fluorescence). This method isn’t as dependable tocollect data, as the efficiency of PCR reactions can decrease during lateramplification cycles as a result of increased accumulation of inhibitors and utilisationof reagents and the data is only gathered after 30-40 cycles of normal PCR. Asa result of calculations happening in ‘real time’, RT-qPCR doesn’t use gelelectrophoresis and thus is an additional adaptation from the preliminaryprocess of PCR. Examples of fluorescent reporter molecules include: doublestranded DNA binding dye or a dye labelled probe. Further adaptations of theprocess include the fact that RT-qPCR can be further separated 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 but 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.

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(Neidler 2017) (AgilentTechnologies 2012)