HighThroughput Droplet Digital PCR Platform for Absolute Quantitation of Nucleic Acids Low Copy Number

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The capability of performing droplet digital PCR (ddPCR) analyses is vital for the NSERC-DG research programs of the applicants. The applicants cannot continue quantifying mRNA, microorganisms and functional genes using "conventional" qPCR technology, and any delays in getting the ddPCR system platform will hamper them from producing the highest quality quantitative results that are expected in the frame of their NSERC research programs. Although real-time PCR (qPCR) can be used for a wide variety of applications, ddPCR is a better choice for many. Due to sample partitioning and absolute digital quantification (instead of standard curve-based relative quantification), ddPCR is best suited for applications requiring high precision, accuracy, sensitivity and reproducibility. Since ddPCR is performed as an endpoint absolute quantification by counting positive droplets, amplification efficiency is not a factor as important as in qPCR and there is no need for an assay-specific standard curve, saving a lot of time and money while preventing skewed results. This unique technology perfectly matches the urgent needs for an in-house absolute nucleic acids quantification system, which will complement the applicants' real-time PCR platforms and will allow them to conduct absolute accurate quantitative nucleic acids analyses. This system is necessary for the advancement and increased efficiency of the environmental toxicology research group at our institute INRS-Institut Armand Frappier. The diverse research activities of the (co)applicants cover the analysis of low-abundance RNA; pathogen detection; viral load detection; miRNA analysis and next generation sequencing sample quantification (all applicants) as well as measurement of plant microbiota (Yergeau), pathogenic bacteria (Déziel), inflammatory and immunologic biomarkers (Bernier), water contamination (Villemur), steroidogenic enzyme (Sanderson) and placental functions (Vaillancourt) affected by environmental factors. Thus, all the applicants' research relies on accurate and reliable quantification of low copy number of nucleic acids in their studied matrices (tissues, bacteria, soil), and thus they are dependent on ddPCR systems. Furthermore, availability of the ddPCR system platform will have a direct impact on HQP research projects, facilitating publication in high-impact journals, and making HQP more competitive in pursuing a research career. A delay in the acquisition of the requested equipment would have a dramatic negative impact on the progress of all the aforementioned research programs and would prevent their completion within the granting period.