Droplet Digital PCR to support basic research

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The applicants of this proposal conduct leading edge basic lipid research in the fields of metabolism, immunology and cell signalling. Studies to date have addressed questions about the influence of fatty acids on whole body physiology, immunology, nutrient metabolism and cell signalling. Studies have used a combination of whole body, tissue and cell culture approaches. Tissues in the body are comprised of a wide range of cell types, which means that we have limited information about the biological role of specific cell types. To address this issue, we recently acquired (with NSERC RTI support) a Bio-Rad flow cytometer with a cell sorter, which can readily obtain rare populations of cells. However, we are not able to perform additional analyses due to the limited amount of sample. This can be overcome by pooling larger numbers of mice, but this is not ideal from an animal welfare perspective and adds significant time and cost. The requested Bio-Rad QX200 Droplet Digital PCR (ddPCR) system has the sensitivity required to detect gene expression changes at the level of the single cell in small samples. This leading-edge technology is only available from Bio-Rad. Competitors are developing competing systems but are not yet on the market. Importantly, the requested ddPCR also has the capability of running standard qPCR analyses, which will address another critical need among the applicants. Currently, they are sharing an Applied Biosystems qPCR machine (7900HT) for routine gene expression analyses but this machine is 15 years old, experiencing repeated failures, and no longer serviceable by the manufacturer (Applied Bio.). Obtaining the ddPCR will support the full range of gene expression analyses required by the applicants in a cost-effective manner. Information by the company indicates that users who are considering the replacement of older qPCR systems are adopting this new platform in increasing numbers in Canada. This is due to competitive pricing and increased capabilities, namely high sensitivity gene expression analysis. Upon acquisition of the ddPCR, Bio-Rad has agreed to provide in-kind on-site training of HQP facilitating the immediate implementation of this equipment. The ddPCR will support three productive and collaborative research programs which are reflected in 22 co-authored publications (last 6 years). The teams commitment to HQP training is also reflected in the training, supervision and mentorship of 137 HQP (73 undergrads, 52 MSc, 7 PhD, 5 Postdocs, last 6 years). It is anticipated that acquiring the ddPCR will enhance training of 40 high quality personnel (per year) across three laboratories, plus additional users (Wright, Simpson, Clark and Bakovic) and their trainees at the University of Guelph. Overall, the ability to analyze changes in gene expression in small samples of highly purified cell types will enable the elucidation of mechanisms of action and enhance the training environment for many high-quality personnel.