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Droplet Digital
™
PCR Applications Guide | 57
6 Gene Expression
Overview
Reverse transcription quantitative PCR (RT-qPCR) is a commonly used method in gene
expression studies. It is straightforward, sensitive, and has a wide dynamic range. There are
two types of approaches for the RT-qPCR reaction: one-step and two-step RT-qPCR.
One-Step RT-qPCR
One-step RT-qPCR simplifies the reaction setup by combining the first-strand cDNA
synthesis (reverse transcription) and qPCR in one mixture. It also greatly reduces the
possibility of contamination by eliminating the cDNA-to-PCR operation step. One-step
RT-qPCR can use only a limited number of probes per sample, but because it amplifies
the whole sample, the sensitivity is greatly enhanced. The disadvantage of one-step
RT-qPCR is that it is less amenable to multiplex assays and allows for less flexibility in
priming strategies.
Two-Step RT-qPCR
Two-step RT-qPCR performs the first-strand cDNA synthesis reverse transcription and
qPCR in separate mixtures. This method allows for the measurement of multiple messages
from a single RNA sample. It also enables you to use different PCR reaction conditions and
priming methods.
To measure gene expression with PCR, you must first convert the RNA into DNA by reverse
transcription. There are three types of primers that may be used in reverse transcription:
■
Oligo(dT) — priming with oligo(dT) results in cDNA synthesis that is biased to the 3' end
of polyadenylated transcripts and will create only cDNA from mRNA templates
■
Random primers — random priming is not subject to end bias and is not limited to
mRNA, but it is sensitive to the sequence and secondary structures of the template
■
Sequence-specific primers — sequence-specific primers can be designed adjacent
to the PCR target, but the ability to perform multiplex qPCR from an individual sample
is limited