Droplet Digital PCR to support basic research

该提案的申请人在代谢，免疫学和细胞信号传导领域进行前沿基础脂质研究。迄今为止的研究已经解决了脂肪酸对全身生理学，免疫学，营养代谢和细胞信号传导的影响问题。研究使用了全身、组织和细胞培养方法的组合。人体组织由多种细胞类型组成，这意味着我们对特定细胞类型的生物学作用的信息有限。为了解决这个问题，我们最近获得了（在NSERC RTI的支持下）一台带有细胞分选仪的Bio-Rad流式细胞仪，它可以很容易地获得稀有细胞群。然而，由于样本量有限，我们无法进行额外的分析。这可以通过汇集更多数量的小鼠来克服，但从动物福利的角度来看，这并不理想，并且增加了大量的时间和成本。所要求的Bio-Rad QX200 Droplet Digital PCR（ddPCR）系统具有在小样本中检测单细胞水平的基因表达变化所需的灵敏度。这种领先的技术只有Bio-Rad提供。竞争对手正在开发竞争系统，但尚未上市。重要的是，所要求的ddPCR还具有运行标准qPCR分析的能力，这将满足申请人的另一个关键需求。目前，他们共用一台Applied Biosystems qPCR机器（7900HT）进行常规基因表达分析，但这台机器已经使用了15年，多次出现故障，制造商（Applied Bio.）不再提供服务。获得ddPCR将以具有成本效益的方式支持申请人所需的全方位基因表达分析。该公司提供的信息表明，正在考虑更换旧qPCR系统的用户正在加拿大越来越多地采用这一新平台。这是由于具有竞争力的价格和增加的能力，即高灵敏度基因表达分析。在获得ddPCR后，Bio-Rad同意为HQP提供实物现场培训，以促进该设备的立即实施。ddPCR将支持三个富有成效的合作研究项目，这些项目反映在22个共同撰写的出版物中（过去6年）。团队对HQP培训的承诺也反映在137名HQP的培训，监督和指导中（73名本科生，52名硕士，7名博士，5名博士后，过去6年）。预计获得ddPCR将加强对三个实验室的40名高素质人员（每年）的培训，加上圭尔夫大学的其他用户（Wright、Simpson、Clark和Bakovic）及其受训人员。总的来说，分析高度纯化细胞类型的小样本中基因表达变化的能力将能够阐明作用机制，并改善许多高素质人员的培训环境。

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.