ShEEP Request for Cellular & Molecular Analysis Core

ShEEP 移动电话请求

• 批准号: 9905897
• 负责人: Jeffrey A. Jones
• 金额: --
• 依托单位: RALPH H JOHNSON VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2020-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9905897
• 关键词: Address; Affect; Agreement; Animal Model; Animals; Area; Biochemical; Biological; Blood; Blood Cells; Caring; Cells; Complement; Copy Number Polymorphism; Core Facility; DNA; Data Quality; Detection; Diagnosis; Disease; Drops; Equipment; Evaluation; Evidence based treatment; Fluorescent Antibody Technique; Functional disorder; Funding; Gel; Gene Expression Profiling; Genetic Diseases; Genotype; Healthcare; Heart Diseases; Human; Image Analysis; Individual; Infrastructure; Injury; Investigation; Laboratories; Maintenance; Medical; Medical center; Mental disorders; Methods; MicroRNAs; Military Personnel; Mission; Modeling; Molecular; Molecular Analysis; Musculoskeletal; Nature; Neurologic; Orthopedics; Pathology; Patients; Proteins; Protocols documentation; RNA; Reproducibility; Research; Research Personnel; Rodent; Sampling; Services; Sheep; Specimen; Spinal cord injury; Stains; Stroke; Techniques; Technology; Time; Tissues; Transcript; Translating; Traumatic Brain Injury; Untranslated RNA; Validation; Vascular Diseases; Veterans; Western Blotting; Work; X-Ray Film; circular RNA; circulating biomarkers; clinical practice; cost; design; digital; experience; fluorescence imaging; human disease; imaging system; instrumentation; next generation sequencing; operation; outcome forecast; programs; tool

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.

该提案满足了增强我们的细胞和分子评估核心（CMEC）的需求， 增加了数字滴式 PCR (ddPCR) 和化学发光/荧光成像分析两种方法； 这些技术将增强我们完成日常工作的能力，以及对血液、细胞、 和组织，作为与我们单独资助的 Bio-Rad 项目相关的共同工作流程的一部分。 ddPCR 和高灵敏度 ChemiDoc MP 成像系统提供最先进的分析， 加快对退伍军人管理局医疗使命重要领域的研究工作，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。 和分子分析工具将补充细胞和分子评估核心的现有技术 位于南卡罗来纳州查尔斯顿的拉尔夫·H·约翰逊退伍军人医疗中心的研究服务中心。 为我们的研究人员提供最先进的设备，以促进综合评估方法 这些核心设施涵盖从亚细胞水平到整个动物的关键设备项目。 通常成本超过单个实验室可以提供的费用，已通过设计建立以支持 并推进我们广泛的研究计划，直接影响退伍军人的医疗保健。 该核心解决的中心假设是，先进技术提供了高度敏感的 在分子水平上评估疾病或损伤的存在和进展，将增强生物 我们的 VA 研究人员所做工作的重要性将通过 4 个主要内容得到解决。 分析目标：1) 高灵敏度检测和绝对定量 DNA 和 RNA 靶标，低至 Bio-Rad 的 ddPCR 的总拷贝数中有 1 个非常适合低拷贝数转录本的研究。 来自尺寸有限（啮齿动物组织等）或丰度非常低的生物样本，以及 对于人类疾病的检测和分析通常至关重要的目标（即检查 循环生物标志物，如微小 RNA、环状 RNA、长非编码 RNA），可用于 诊断和预后；2）通用的标准DNA和RNA分析技术 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） 3) 蛋白质印迹上蛋白质条带的可重复定量和标准化（以 总蛋白（采用 Bio-Rad 泳道免染色凝胶技术），使用标准化学发光方案和 高灵敏度成像系统，代替 X 射线胶片；以及 4) 多重荧光抗体检测，在 通过蛋白质印迹同时鉴定多种分析物，这不仅缩短了获得结果的时间， 考虑到我们核心设施的多功能性和集成性，VA 也受到关注。 研究人员将能够从“日常”和先进的技术协议中获得高质量的数据 可扩展的方式，以促进血液、细胞和组织作为关键成分的生化分析 他们资助的研究结果是直接从 DNA、RNA 和蛋白质检查中获得的。 适用于影响我们退伍军人的核心医学问题的病理生理学，例如血管疾病， 心脏病、神经系统疾病、肌肉骨骼疾病、中风、创伤性脑损伤和脊髓 拟议设备与我们现有的细胞和分子评估核心的集成将填补损伤。 技术差距，使我们的研究人员能够对其样本进行全方位的生化评估。 代表了一个独特的基础设施，将显着推进我们当地正在进行的研究 VAMC 所要求的仪器将进一步增强当地 VA 研究能力。 退伍军人事务部部长卓越蓝图中概述的目标。