Article 
Not All PCR is Created Equal
Real-time qPCR is a highly sensitive technique that has become the tool of choice for the rapid, sensitive quantification of nucleic acid in various biological samples and has been used in gene expression analyses for more than 35 years.5 It has a large dynamic range allowing for quantification of both low-and high-abundance targets, and can discriminate splice variants. In addition, it is easily automated, facilitates multiplexing, and is extremely economical. However, RT-qPCR can only detect changes in gene expression greater than twofold, provides primarily relative quantification of RNA transcripts, and data accuracy is extremely reliant on the amplification efficiency of the PCR reaction.
Digital PCR, developed in the early 90s, 7 is an alternative, albeit less adopted method, to RT-qPCR that can address some of the challenges faced by the classic approach to measuring gene expression. Because each sample is partitioned into thousands of nano reactions, this method provides absolute quantification of RNA transcripts and doesn’t necessitate the creation of standard curves to quantify RNA or PCR efficiency. It is also more sensitive than RT-qPCR, capable of detecting changes in expression as low as 10–30%, and can detect rare transcripts from complex backgrounds.8 Digital PCR is also less affected by the presence of contaminants and PCR inhibitors in the sample.
Advantages and Disadvantages of RT-qPCR and Digital PCR
| RT-qPCR | Digital PCR | |
|---|---|---|
| Advantages | Industry standard/established method, economical, high throughput, easy to automate, broad dynamic range, detects changes in gene expression as low as twofold | Robust to contamination, detects rare transcripts in complex backgrounds, absolute quantification, detects changes in gene expression less than twofold |
| Disadvantages | Sensitive to contamination, requires a reference standard and standard curves, relative quantification, variability, dependence on amplification efficiency | Less broad dynamic range, limited availability of personnel knowledgeable in the technique |
The Pros and Cons of RT-qPCR and Digital PCR Across Therapeutic Development
Early Discovery
The first stage of therapeutic development is target identification, which largely occurs in the academic research laboratory9 where critical basic information about the disease's molecular mechanisms are elucidated. Potential therapeutic “targets” are typically identified by cataloging differences in gene expression between groups of samples (typically, healthy versus diseased) and then selecting differentially expressed genes for further analysis.
While microarrays or RNA-Seq are typically used at this stage, both qPCR and digital PCR can be effective approaches for identifying targets. Many academic researchers, for example, are in niche fields that may only require analysis of 20 or 30 potential targets with known sequences, making microarrays or RNA-Seq unnecessary. However, PCR techniques do play an essential role in validating potential targets identified via microarray and RNA-Seq experiments. RT-qPCR can be used when a broad dynamic range is necessary, while digital PCR is useful for validating rare transcripts with subtle gene expression changes.
After validating molecular targets, candidate therapeutic molecules are tested to determine whether they elicit the desired effect on the target (for example, does a candidate therapeutic molecule repress transcription of a gene upregulated in lung cancer?). Often, this involves screening large numbers of molecules against genetically modified cell lines expressing the target(s) of interest. Both RT-qPCR and digital PCR are useful for confirming desired gene modifications in those cell lines; however, digital PCR can detect gene modifications/edits more quickly and at an earlier time point in cellular growth,10 reducing time and other resources spent on propagating cell lines that don’t have the desired edits. RT-qPCR and digital PCR can also rapidly assess the impact of the various candidate therapeutic molecules on gene expression.
Deeper characterization and optimization of candidate therapeutic molecules also require the development of custom cell lines (as well as analysis of gene expression by those cell lines in response to treatment). At this stage of therapeutic development, scale-up is critical, so the early validation of cell lines with the desired genetics enabled by digital PCR makes it a great choice.
Preclinical Studies and Manufacture
The most promising therapeutic candidates move on to preclinical studies, where toxicity and biodistribution are carefully characterized. Mistakes here could lead to disastrous consequences in clinical trials, so sensitivity and reproducibility are critical. Using qPCR is ideal when analyzing biodistribution samples because of its wider dynamic range, high throughput, and short time to results. Digital PCR is more reproducible and more sensitive than qPCR for rare targets in limited samples; however, the sensitivity of qPCR can be enhanced by increasing input sample and/or reaction volume. Additionally, animal models are often used at this stage, so samples can be limited — another situation in which digital PCR excels. Although currently, digital PCR isn’t widely used in the pharmacology phases of therapeutic development, this is a great opportunity area for the technique, and its use is on the rise.
Once a candidate therapeutic proceeds through clinical trials, rapid, efficient, large-scale manufacture of the therapeutic is necessary to achieve regular clinical use. Process development is thus a critical component of any therapeutic discovery and development effort moving toward completion. Potency testing of therapeutic lots is a particularly required stage in developing the final pharmaceutical product. It cannot be released if the final product does not meet the required potency. In many cases, cell-based assays are used to confirm potency, but in cell and gene therapies, multiple assays are needed to validate a therapeutic’s potency. For instance, therapies based on the adeno-associated virus (AAV) often require potency testing using cell-based assays and in vitro methods. Alternative assays, using a combination of RT-qPCR and standard protein assays, have also proven to be highly effective at analyzing potency in samples from AAV gene therapy vectors.11
PCR is a Critical Method Used in Therapeutic Development
PCR techniques have and will continue to play a critical role in supporting gene expression analyses throughout the therapeutic development process due to their ease of use, sensitivity, amenability to automation, efficient scale-up, and cost-effectiveness. Investigators in the therapeutic discovery and development space can select from two powerful PCR techniques — qPCR and digital PCR — to address their sensitivity, throughput, and scale-up needs at various stages of therapeutic development, from early discovery to manufacture.
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