High throughput digital PCR facility

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The equipment requested in this proposal is essential to researchers at the University of Guelph who are interested in quantifying the expression level of specific genes that are present in very low levels or found only in a few cells. The requested funds will build a high throughput facility that will allow for genetic material (DNA, mRNA, rRNA) to be efficiently extracted using a Precellys24-Cryolys refrigerated homogenizer and then quantified using a BioRad digital droplet polymerase chain reaction (PCR) instrument. The Precellys24-Cryolys uses microbeads in an optimally temperature-controlled environment to grind, lyse and extract mRNA from a broad range of tissues (plant and animal) resulting in greater quality and yield of genetic material per extraction than the manual methods currently in use. As such, access to this equipment will also allow us to significantly reduce the amount of biological material needed for each experiment. This tool will also dramatically increase the rate of sample preparation and therefore remove a bottleneck in data collection. The requested BioRad instrument uses droplet partitioning to preform thousands of independent PCR reactions in the same sample. As a result it provides an absolute measure of target DNA molecules. This eliminates the need for stably expressed reporter housekeeping (RH) genes, and reduces the effects of target competition allowing differentiation of transcripts that differ by a single nucleotide. The use of RH genes is a requirement of currently used relative quantification methods (real-time PCR) and has had a marked impact on a number of projects in the applicants’ labs. These examine the molecular regulation of development and how this is impaired by environmental stressors. The requested equipment will enable the applicants to examine a range of biological phenomena. This includes determining how changes in environmental conditions and environmental contaminants affect the health and reproductive capacity of freshwater fishes, characterizing how the heart remodels in response to a physiological stressor, and examining how the number of a specific gene in a nucleus influences the evolution of an inheritable trait. These instruments will be located in the centralized Genomics Facility at the University of Guelph and will ultimately be accessible by researchers across campus. There are over 50 labs on campus that utilize the existing gene quantification instrumentation of the Genomics facility and who could legitimately exploit the enhanced capabilities of the digital droplet gene quantification system.