ShEEP Request for Cellular & Molecular Analysis Core

Ralph H Johnson Va Medical Center, Charleston SC

Investigators

Abstract

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.