High throughput digital PCR facility

这项建议中所要求的设备对于圭尔夫大学的研究人员来说是必不可少的，他们有兴趣量化存在于非常低水平或仅在少数细胞中发现的特定基因的表达水平。申请的资金将建造一个高通量设施，使遗传物质(DNA、mRNA、rRNA)能够使用Precellys24-Cryolys冷藏均质机有效提取，然后使用BioRad数字液滴聚合酶链式反应(PCR)仪器进行定量。Precellys24-Cryolys使用微珠在最佳的温度控制环境中研磨、裂解和从广泛的组织(植物和动物)中提取mRNA，从而使每次提取的遗传物质的质量和产量都比目前使用的人工方法更高。因此，使用这种设备还将使我们能够显著减少每次实验所需的生物材料的数量。这一工具还将极大地提高样品准备的速度，从而消除数据收集中的瓶颈。所要求的BioRad仪器使用液滴分配在同一样本中进行数千个独立的聚合酶链式反应。因此，它提供了对目标DNA分子的绝对测量。这消除了对稳定表达的报告管家(RH)基因的需要，并减少了靶标竞争的影响，从而允许区分不同于单个核苷酸的转录本。RH基因的使用是目前使用的相对定量方法(实时聚合酶链式反应)的要求，并对申请者实验室的一些项目产生了显著影响。这些研究考察了发育的分子调控以及环境应激源是如何损害这一调控的。所要求的设备将使申请者能够检查一系列生物现象。这包括确定环境条件和环境污染物的变化如何影响淡水鱼的健康和繁殖能力，确定心脏如何对生理应激源做出反应，以及检查细胞核中特定基因的数量如何影响可遗传特征的进化。这些仪器将放置在圭尔夫大学的集中基因组学设施中，最终将由校园内的研究人员使用。校园内有50多个实验室利用基因组学设施现有的基因量化仪器，他们可以合法地利用数字水滴基因量化系统的增强能力。

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.