DURIP Digital Polymerase Chain Reaction (PCR) apparatus for ONR funded research
Abstract
This proposal requests funds to purchase of an Applied Biosystems Digital Polymerase Chain Reaction (PCR) apparatus to expand our Office of Naval Research (ONR) funded research capability focused on the role of extracellular vesicles (EVs) in decompression sickness (DCS). EVs are lipid bilayer-enclosed sub-cellular structures that increase in association with inflammation. They play roles in cell-to-cell communication due to their surface structure and content of a variety of inflammatory mediators, enzymes or organelles that generate free radicals and nucleic acids. To date, our studies of EVs have focused on improving isolation techniques, establishing their relationship with DCS in humans and animal models, determining EVs cells of origin and their inflammatory protein cargo. Acquisition of Digital PCR apparatus will provide state-of-the-art capability to include nucleic acid analysis in our investigations.--DCS presents a health risk for all deep-sea divers and it poses a constraint to provocative diving performed by Navy personnel and for submarine escape. The central place of bubbles as an inciting agent for DCS is widely accepted. However, decompression generates asymptomatic blood-borne bubbles indicating that additional factors are at play for precipitating tissue injuries. We have been continuously funded by ONR to study the roles of EVs in DCS since 2010 and have published 34 peer reviewed papers. We have demonstrated that EVs increase in proportion to the provocative nature of a diving profile and are capable of causing extensive tissue damage.--We are seeking funds to enhance our ability to assess the nucleic acid cargo of EVs. We are especially interested in improving our ability to detect what are called microRNA (miRNA) species, as a growing body of work has implicated their role in EVs biology. MicroRNAs are short (19#25 nucleotides), single stranded and nonprotein-coding RNAs that regulate gene expression by binding to the 30-untranslated region of a target messenger RNA (mRNA). A single miRNA species can bind to many different mRNA targets causing either increased or decreased production of proteins and thus perturb diverse biological processes. Altered miRNA profiles occur in many disorders, influence disease pathogenesis, and sometimes are used as biomarkers for diagnosis and prognosis. RNA-sequencing of asymptomatic, health divers as well as those with DCS has identified transient enrichment of transcripts involved in acute inflammation, activation of innate immunity and free radical scavenging pathways, and specific upregulation of transcripts related to neutrophil function and degranulation.- -A major problem with miRNA analysis is their relatively rare presence in alternative EVs sub-groups. Both traditional real-time or quantitative PCR (qPCR) and newer technology digital PCR identify and quantify nucleic acids in a sample by amplifying a target nucleic acid molecule with a DNA polymerase enzyme. Traditional qPCR has the advantage of a broad detection range when targets are unknown, whereas digital PCR has greater sensitivity. The Applied Biosystems apparatus combines both functional capabilities and thus has greater flexibility as well as time and resource savings. Digital PCR provides a linear response to the number of copies present to allow for small fold-change differences to be detected (qPCR records data in the exponential phase of PCR amplification and requires a minimum of 2-fold difference in copy number). Digital PCR also provides a quantitative data output without reliance on references or standards for conversion of data points. Single-molecule resolution interrogation enables identification and quantification of molecules containing multiple targets (e.g. phased targets as occur with miRNA). Digital PCR exceeds the technical capability present in our institutional PCR core facility. This instrument will also improve our ability to educate future scientists through DoD research.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jun 13, 2024
- Source ID
- N000142412253
Entities
People
- Stephen R. Thom
Organizations
- Office of Naval Research
- United States Navy
- University of Maryland