APR January/February 2022 - 74

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INSTRUMENTATION
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One-Step Versus Two-Step Methods
for RNA Detection
Figure 2. SYBR and TaqMan PCR detection technologies.
F: fluorescent molecule, Q: quencher
Figure created with Biorender.com
and fluorescent signal. Several technologies are available to improve
primer/probe performance such as the addition of a minor groove
binder (MGB) to increase melting temperature, enabling shorter, more
target-specific probes, or an additional internal quencher to reduce
background signal.
While SYBR is lower in cost, TaqMan has a higher level of specificity
and sensitivity, and allows for duplexing or multiplexing. SYBR binds
non-specifically to all dsDNA, so false positive signals are possible via
primer dimers or other non-target DNA.1,2
qPCR Versus dPCR
The two main platforms utilized for PCR-based quantitative methods
are quantitative real-time PCR (qPCR) and digital PCR (dPCR). In qPCR
each reaction well is a single fluorescent signal that is proportional to
the amount of starting target DNA present in the reaction mixture. On
the other hand, dPCR employs the separation of the reaction mixture
and sample into thousands of partitions. Specifically, in droplet digital
PCR (ddPCR), these partitions are water-in-oil droplets. Based on a
fluorescence amplitude threshold, droplets can be scored as positive
or negative for the PCR. Using a Poisson distribution calculation, the
proportion of positive and negative droplets can then be used to
determine absolute concentration of the target DNA sequence.
Although qPCR has the advantage of being a less expensive PCR
method and it can be miniaturized to 384-well, ddPCR has several
advantages to qPCR including that it does not require a standard curve,
is less influenced by PCR bias because of matrix interference or other
PCR inefficiencies, and has a high level of sensitivity and precision.
Therefore, ddPCR is the preferred method for absolute quantitation
of in-process intermediates that may have different buffer and matrix
compositions.3
It also has become the platform of choice for many
applications in gene therapy.3,4,5
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RNA levels can be measured by isolating total RNA and reverse
transcribing it to cDNA. The resulting cDNA is quantified via PCR.
Depending on the type of target to be measured, different reverse
transcription (RT) and PCR protocols can be used. RT-qPCR and RTddPCR
methods may utilize either a one-step method, whereby the
reverse transcription and subsequent qPCR or ddPCR occur in the same
reaction mix, or two-step where the cDNA is generated in a reverse
transcription reaction and then subsequently quantified by qPCR
or ddPCR. One-step PCR methods typically use the reverse primer
from the detection primer/probe set for the RT step. This method
reduces the number of steps required to quantitate RNA expression
and involves less sample handling and pipetting steps, thus reducing
assay variability and possibilities for sample contamination. One
caveat is that one-step methods do not generate a stock of cDNA
for later use, preventing potential for re-analysis. One-step methods
also limit the ability to separately optimize the RT and PCR reactions.
Although two-step PCR methods require an additional step, they
allow for a stock of cDNA to be generated for multiple PCR reactions.
This can be useful for method troubleshooting as well as allowing
both steps to be fully optimized independently. Two-step methods
can employ several different types of primer for the RT step including
the reverse primer from the detection primer/probe set, oligo(dT)
primers, or random primers.
For qPCR methods, with low starting material or low abundant targets,
two-step methods can provide better sensitivity and more accurate
quantitation due to the ability to titrate cDNA levels for the PCR
reaction. This allows for diluting PCR inhibitors that may be carried
over from the RNA isolation and RT reaction. However, for ddPCR
one-step and two-step methods are less prone to influence by matrix
effects and have similar sensitivity.4
Characterization of AAV
Using PCR Technologies
AAV gene therapy analytical control
strategies necessitate
characterization of the AAV at multiple stages throughout the
transduction pathway. Below we discuss how PCR-technologies can
be utilized for many aspects of this characterization for both in vitro
and in vivo studies (Figure 1).
AAV Genome Titer
One of the most important analyses performed for AAV vectors is
determining the physical quantity as represented by genome titer.
AAV genome titer is used to define dosing in preclinical animal
models as well as in patients in the clinic. Additionally, genome
titer is critical to support efforts of process development groups, to
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APR January/February 2022

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