Extracellular Vesicles in Saliva as Biomarkers of Exposure and Effect

Abstract

TPS 651: Air pollution exposure modeling 1, Exhibition Hall, Ground floor, August 27, 2019, 3:00 PM - 4:30 PM Background: Saliva collection is fast, easy, inexpensive, and non-invasive and saliva is easy to transport, making it an ideal biofluid for epidemiological research. However, there is a relatively low concentration of biomarkers in saliva when compared to other biofluids. Thus, successful measurement of salivary analytes such as extracellular vesicle (EV) microRNA requires optimal collection, processing, and storage procedures. Here, we demonstrate the feasibility of collecting multiple saliva samples from participants in field research and show that we can obtain high-quality molecular biomarkers such as EV microRNA. Methods: Subjects refrained from eating, drinking, or oral hygiene for at least one hour prior to collection. Samples were then collected by passive drool. Saliva was kept on ice and processed within one hour of collection. After removing cells and debris, EVs were isolated from cell-free saliva supernatant using an exosome precipitation solution. Re-suspended EVs underwent nanoparticle tracking analysis to assess EV size and concentration. EV total RNA was extracted and RNA quality and yield were assessed using a BioAnalyzer (Agilent). Results: In an initial pilot, we collected five samples from two individuals. Extracted saliva EVs contained up to 90% microRNA. Across participants, saliva EVs had an average concentration of 1.5E+10 particles/mL, with an average size of 363nm (sd: 191nm). Exploratory analyses examining the association between microRNA concentration and personal exposure measurements taken while bicycling outdoors (e.g. particulate matter dose, black carbon dose) suggest that there is a positive association between air pollution exposure while bicycling and saliva EV microRNA concentration. Conclusion: Low volume starting inputs (500uL-1mL) of whole saliva can yield high quality salivary EV microRNA in quantities sufficient for downstream applications such as RNA sequencing. Subsequent analysis of microRNA expression profiles and changes in microRNA levels in saliva may lead to biomarkers of exposure to environmental toxicants.