ShEEP Request for Cellular & Molecular Analysis Core

该提案解决了增强我们的细胞和分子评估核心（CMEC）的需求， 增加了数字滴加PCR（ddPCR）和化学发光/荧光成像分析; 这些技术将提高我们完成日常工作的能力，以及对血液，细胞， 和组织，作为与我们个人资助的项目相关的共同工作流程的一部分。伯乐公司 ddPCR和高灵敏度的ChemiDoc MP成像系统，提供最先进的分析， 加快对退伍军人事务部医疗使命重要的领域的研究工作，包括精神健康障碍， 创伤性脑损伤、脊髓损伤、心脏病、骨科损伤和中风。此外，这些细胞 和分子分析工具将补充细胞和分子评估核心中的现有技术 位于Ralph H.位于南卡罗来纳州查尔斯顿的约翰逊退伍军人医疗中心。我们的目标是 为我们的调查人员提供最先进的设备，以促进全面的评估方法 从亚细胞水平到整个动物。这些核心设施，围绕关键设备项目而建 通常成本超过一个实验室所能提供的，已经通过设计建立，以支持 并推进我们广泛的研究计划，直接影响我们的退伍军人的医疗保健。的 该核心所解决的中心假设是，先进的技术提供了一个高度敏感的 在分子水平上评估疾病或损伤的存在和进展，将增强生物学特性， 我们的VA调查员正在做的工作的重要性。这一假设将通过4个主要 分析目的：1）高灵敏度检测和绝对定量的DNA和RNA目标低至 1/100万份。Bio-Rad的ddPCR非常适合研究低拷贝数转录本 从尺寸有限的生物样本（啮齿动物组织等），或以非常低的丰度存在， 代表对于人类疾病的检测和分析（即， 循环生物标志物，如微RNA、环状RNA、长非编码RNA），并且其可用于 诊断和预后; 2）标准DNA和RNA分析技术通常用于多功能和 可扩展的工作流程（即基因分型、基因表达分析、拷贝数变异和下一代- 测序验证）; 3）蛋白质印迹上蛋白质条带的可再现定量和标准化（以 用Bio-Rad免染色凝胶技术测定每泳道总蛋白质），使用标准化学发光方案， 高灵敏度的成像系统，代替X射线胶片;以及4）多重荧光抗体检测， 努力通过蛋白质印迹法同时鉴定多种分析物。这不仅缩短了取得成果的时间， 而且取决于消耗品的使用。鉴于我们核心设施的多功能性和综合性，VA 调查人员将能够获得高质量的数据，从“每一天”和先进的技术协议，在一个 可扩展的方式，以便于血液，细胞和组织的生化分析作为一个关键组成部分 他们资助的研究。从DNA、RNA和蛋白质的检测中获得的结果直接 适用于影响我们退伍军人的核心医学问题的病理生理学，如血管疾病， 心脏病、神经系统疾病、肌肉骨骼病变、中风、创伤性脑损伤和脊髓 损伤建议的设备与我们现有的细胞和分子评价核心的整合将填补 技术差距，使我们的研究人员能够对他们的样本进行全方位的生化评估。这 代表了一个独特的基础设施，将大大推进我们当地正在进行的研究， VAMC。所要求的仪器将进一步提高当地VA研究能力， 在退伍军人事务部卓越蓝图秘书概述的目标。

This proposal addresses a need for the enhancement of our Cell and Molecular Evaluation Core (CMEC) with the addition of digital drop PCR (ddPCR) and chemiluminescence/fluorescence imaging analysis; two technologies that will enhance our ability to complete every day, as well as, advanced analyses of blood, cells, and tissues, as a part of the common work flow associated with our individually funded projects. Bio-Rad's ddPCR and highly sensitive ChemiDoc MP imaging systems, provide state-of-the-art analyses that will accelerate research efforts in areas important to the VA medical mission including mental health disorders, traumatic brain injury, spinal cord injury, cardiac disease, orthopedic injury, and stroke. Furthermore, these cell and molecular analysis tools will complement existing technology in the Cell and Molecular Evaluation Core housed within the Research Service at the Ralph H. Johnson VA Medical Center in Charleston, SC. We aim to provide state-of-the-art equipment to our investigators, in order to facilitate comprehensive methods of evaluation spanning from the subcellular level to the whole animal. These core facilities, built around key equipment items often costing more than what a single laboratory can provide, have been established by design in order to support and advance our wide-ranging research program that directly impacts the health care of our Veterans. The central hypothesis addressed by this core, is that advanced technology that provides a highly sensitive assessment of disease or injury presence and progression at the molecular level, will enhance the biological significance of the work being done by our VA investigators. This hypothesis will be addressed through 4 Primary Analytical Objectives: 1) Highly sensitive detection and absolute quantitation of DNA and RNA targets as low as 1 out of 1,000,000 total copies. Bio-Rad's ddPCR is highly suited for the study of low copy number transcripts from biological specimens that are limited in size (rodent tissues, etc.), or exist at very low abundance, and represent targets that are often critical for the detection and analysis of human disease (i.e. the examination of circulating biomarkers like micro-RNAs, circular-RNAs, long non-coding-RNAs) and that can be used for diagnosis and prognosis; 2) Standard DNA and RNA analytical techniques commonly used in a versatile and scalable workflow (i.e. genotyping, gene expression analysis, copy number variation, and Next-Generation- Sequencing validation); 3) Reproducible quantitation and normalization of protein bands on Western blots (to total protein per lane with Bio-Rad stain-free gel technology) using standard chemiluminescence protocols and a highly sensitive imaging system, in place of x-ray film; and 4) Multiplex fluorescent antibodies detection, in an effort to identify multiple analytes simultaneously by western blotting. This not only cuts down on time-to-results, but also on usage of consumables. Given the versatility and integrated nature of our core facilities, VA Investigators will be able obtain high-quality data, from “every-day” and advanced technical protocols, in a scalable fashion, in order to facilitate the biochemical analysis of blood, cells, and tissues as a critical component of their funded studies. The results obtained from the examination of DNA, RNA, and protein, are directly applicable to the pathophysiology of the core medical issues affecting our Veterans such as vascular disease, cardiac disease, neurologic illness, musculoskeletal pathologies, stroke, traumatic brain injury, and spinal cord injury. The integration of the proposed equipment with our existing Cell and Molecular Evaluation Core will fill technical gaps and allow our researchers to conduct full-service biochemical assessments of their samples. This represents a unique infrastructure that will significantly advance the research being conducted at our local VAMC. The requested instrumentation will further enhance local VA research capabilities in line with the objectives outlined within the Secretary of the Department of Veterans Affairs Blueprint for Excellence.