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PCR: The "Workhorse" Technology

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Polymerase chain reaction (PCR) is regarded as one of the biotechnological breakthroughs of the late twentieth century that continues to play an integral role in scientific research across a wide variety of disciplines.

In this interview, Technology Networks spoke with Dr Jan Hellemans, CTO and co-founder of CRO Biogazelle, as well as co-author of the MIQE guidelines, to learn why PCR-based technologies are the "workhorse" technologies for the company. Dr Hellemans also outlines specific research project examples for which PCR has been instrumental, such as immuno-oncology studies and COVID-19, and discusses jow PCR complements other technologies in Biogazelle's portfolio.

Molly Campbell (MC): For our readers that may be unfamiliar, please can you discuss the work Biogazelle conducts as a CRO, and why PCR technologies are a valuable tool for Biogazelle?

Jan Hellemans (JH):
Biogazelle is a CRO specializing in high-value applications to support pharmaceutical research, clinical trials and diagnostic test development. To accelerate the development of small molecules, RNA targeted drugs and adoptive cell therapies, we apply a suite of genomic and transcriptomic technologies, to find and validate RNA biomarkers, and to assess efficacy, safety and toxicity.

RNA sequencing workflows are typically used in the discovery phase of projects, using precious clinical samples to identify candidate biomarkers. The workhorse technologies in our lab are, however, quantitative PCR and droplet digital PCR (ddPCR). They are used to support all research as well as all measurements of specific genes or other targeted analyses.

MC: There are a number of different types of PCR technologies available. Can you discuss why you specifically adopt ddPCR and qPCR?

For me, selecting a technology is not about pinpointing a winner. It is about understanding the intrinsic merits, challenges and limitations of the different technologies available on the market or developed in house. Having said that, PCR is clearly a corner stone technology in most if not all molecular labs. The type of analyses we are doing for our clients mostly involve quantification of certain sequences, be it the expression of a gene, the copy number of a transgene or the viral load. qPCR and ddPCR are clearly the go to technologies for quantification of a selected and limited number of targets.

MC: Are you able to discuss examples of projects that you have undertaken using ddPCR and qPCR?

The scope of projects in which we apply qPCR or ddPCR is quite broad. At the start of the COVID-19 crisis, the Belgian government requested Biogazelle to leverage its capabilities and provide testing capacity to support the national screening needs. Our expertise in qPCR assay validation and instrumentation for high-throughput testing, allowed us to act quickly and take over a sizable part of the national testing capacity. In view of the need for high-throughput test capacity as well as for reliable and sensitive detection of SARS-CoV-2, qPCR was the clear technology of choice.

Immuno-oncology is a field that has witnessed an explosion of interest in recent years and a large number of clinical trials are ongoing in this space. As adoptive cell therapies commonly utilize viral vectors for transfection, there is a need to verify the absence of replication competent lenti- or retro-viruses and to monitor the vector copy number (a measure for the persistence of CAR-T cells in the body). Both require a sensitive and accurate quantification of the respective DNA sequences. In practice, both qPCR and ddPCR are being used, the choice being dependent on the particular requirements of the sponsor.

Towards the earlier phases of drug discovery, we have developed a unique expertise in screening antisense oligonucleotides for their potential to specifically reduce the expression of their target gene. For these, high throughput screening methods have been developed for which tens of thousands of qPCR reactions have to be performed for a single screening. Also, large sample numbers and a small number of genes of interest are a perfect match for qPCR’s strengths.

MC: Please can you talk to us about how technologies such as qPCR and RNA-Seq are co-existing in your portfolio?

As stated before, every technology has its place. The research question dictates the best fit methodology. For the quantification of large numbers of sequences, RNA-seq is typically the go to technology. For more targeted analysis, qPCR and ddPCR, because of their lower cost and larger sample throughput, are better suited. As a general rule of thumb, RNA-seq could be considered an early-stage discovery tool whereas qPCR and ddPCR would be a better match for later stage confirmation or testing needs.

MC: What are the biggest challenges that you encounter as a CRO, and how do you work to overcome them?

Biogazelle is a specialty service provider. This means we typically cannot rely on off-the-shelf solutions but have to develop tailor made assays and methods. Combining flexibility and scientific excellence with the quality assurance needed for most of our clients is a significant challenge. Working together on a single project across different departments (science and QM, lab and data analysis) and training of people to understand these needs is how we can make this possible.

MC: PCR has proven an instrumental tool in COVID-19 testing. Please can you tell us about Biogazelle's work in this space? How have you ensured efficiency and accuracy is achieved in the testing process?

It is important to clarify that accuracy in the context of SARS-CoV-2 testing refers to sensitivity and not quantification; we’re not interested in quantifying with precision a patient’s viral load, but to establish whether the virus is present or not. From that perspective, we need to be able to detect low viral counts since patients in the early days of infection may have a low viral load but will inevitably become transmitters of the virus later on. We dedicated a lot of effort to optimize extraction procedures and detection methods to improve test sensitivity. During this process, aspects such as availability of instruments and reagents as well as overall test efficiency had to be considered. Doing this all under immense time pressure was one of the biggest challenges we ever faced, but one which I am proud to say we managed to overcome.

Jan Hellemans was speaking to Molly Campbell, Science Writer, Technology Networks.

ddPCR is an innovative technology available from Bio-Rad. If you’re interested in finding out how other experts are utilising it in their research, and what ddPCR systems are available, please browse here