Quantitative sort of RNA that is used and

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reverse transcription PCR (RT-qPCR) is an alteration of the normal PCR process
and is used in many applications such as gene expression analysis, pathogen
detection, genetic testing and disease research. (ThermoFisher 2017) PCR uses double stranded DNA whereas RT-qPCR
utilises the use of RNA (either as pre-mRNA or as purified mRNA). Hence, this
is an adaptation of the original PCR process, which is otherwise restricted to the
use of double stranded DNA. As RT-qPCR utilises RNA, it entails an additional
step, where the RNA is reverse transcribed by reverse transcriptase to form
cDNA. A primer is needed to allow the reverse transcription of RNA and the
primer is chosen based on the sort of RNA that is used and the length of the
RNA that 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 of
mRNA; Random primers – a stretch of 6-9 bases, which bind at various points
along the stretch of RNA; Sequence specific primers – are primers which target
specific mRNA sequences. This is in supplement to the use of primers, in the
PCR process of both normal PCR and RT-qPCR, hence is a further adaptation of
the process. The progress of the amplification reaction in RT-qPCR is observed
using a fluorescent reporter molecule allowing calculation of the initial
template quantity in ‘real time’ as the PCR progresses, as opposed to normal
PCR where data collection occurs at the end of the reaction (calculations are
made based on the final fluorescence). This method isn’t as dependable to
collect data, as the efficiency of PCR reactions can decrease during later
amplification cycles as a result of increased accumulation of inhibitors and
consumption of reagents and the data is only collected after 30-40 cycles of
normal PCR. As a result of calculations happening in ‘real time’, RT-qPCR
doesn’t use gel electrophoresis and thus is an additional adaptation from the
initial process of PCR. Examples of fluorescent reporter molecules include:
double stranded DNA binding dye or a dye labelled probe. Further adaptations of
the process include the fact that RT-qPCR can be further separated into either
a one step process or a two-step process: the difference between the two being
that in the one step process, both the reverse transcription and PCR occur in
the same test tube whereas in the two-step process, the two steps occur in two
separate test tubes. The actual three step process of PCR remains the same in
both RT-qPCR and PCR. (Neidler 2017) (Agilent
Technologies 2012)


Categories: Applications


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