ABSTRACT
Advancing biomedical studies necessitates the development of cutting-edge technologies for the rapid extraction of nucleic acid. We characterized an RNA capture pin (RCP) tool that is non-destructive to the sample and enables rapid purification and enrichment of mRNA for subsequent genetic analysis. At the core of this technology is a pin (200 µm × 3 cm) functionalized with dT15 capture sequences that hybridize to mRNA within 2 min of insertion in the specimen. Two methods for immobilizing the oligos on the surface of the RCPs were investigated: gold-thiol and biotin-streptavidin. The RNA capture efficiency of the RCPs was assessed using a radish plant. The average reverse transcription-quantitative polymerase chain reaction (RT-qPCR) cycle amplification values were 19.93 and 24.84 for gold- and streptavidin-coated pins, respectively. The amount of RNA present on the surface of the probes was measured using the Agilent 2100 Bioanalyzer. RNA sequencing was performed to determine the mRNA selectivity of the RNA capture pin. Gene read count analysis confirmed that the RNA purified via the gold-plated RCPs contained 70% messenger RNA, 10% ribosomal RNA, and 20% non-coding RNA. The long-term stability of the bond between the dT15 oligos and the surface of the RCPs was assessed over 4 months. A significant decrease in the dT15 surface coverage of the streptavidin-coated RCPs was observed after 2 weeks of storage at 4 °C. The gold-thiol RNA capture pins exhibited a retention rate of 40% of the oligos after 4 months of storage.
ABSTRACT
Space missions pose threats to the health of the astronauts due to long-term exposure to galactic cosmic rays and solar particle events comprised predominantly of medium to high energy protons, energetic helium ions, and energetic high atomic number particles (HZEs). While the tissue-specific effects of radiation have been studied extensively, the changes in exosomal miRNA expression levels in response to acute radiation exposure have not been assessed. Extracellular vesicles (EVs) originate from the host cells and contain nucleic acid and proteins that can modify the physiology of the receiving cells via the transfer of genomic, proteomic, and lipids cargo. Detection and analysis of miRNA cargo of circulating EVs is an emerging method for non-invasive diagnosis and monitoring of neurological disorders. This study characterizes the EV-derived miRNA expression profiles of human astrocytes to identify those that are altered after treatment with 3 Gy proton radiation as biomarkers of neurological radiation injury. The relationship between radiation and miRNA extracellular vesicles expression levels was investigated in human astrocytes after treatment with 3 Gy proton radiation at Willis-Knighton Cancer Center. Microarray analysis was performed using miRNA from the EVs enriched fraction in the cell culture medium collected from sham-control and radiation-treated cells. The exosomal levels of 13 miRNAs were significantly (FDR p < 0.05) down-regulated after exposure to high-energy radiation. The computational analysis identified hsa-miR-762, hsa-let-7c-5p, and has-let-7b-5p regulate the highest number of genes being associated with cognitive, mental, and motor delay. These miRNAs target the same subset of genes (Amd1, CCNF, COX6B, PLXND1) that are associated with epileptic encephalopathy; frontotemporal dementia; mitochondrial complex iv deficiency, and a rare neurological condition (Moebius syndrome) respectively. GO enrichment analysis of the biological processes identified overrepresentation in mRNA polyadenylation and regulation of glutamine and long fatty acids transport. Gene expression analysis confirmed the upregulation of the glutamine synthetase after irradiation. Significant fold enrichment of GO l-glutamine transmembrane transporter activity was identified in the molecular function category as well indicating exosome-mediated regulation of this important pathway after proton radiation exposure.
