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The discovery of extracellular RNAs (exRNAs) in the bloodstream has raised intriguing questions about whether regulatory RNA molecules, either endogenously produced or taken up from our environment (e.g., through diet), can influence our physiology by acting as blood-borne hormones. Although we and others have studied circulating exRNAs for several years now, the identity of >95% of exRNA species in blood and other body fluids remains a mystery. Furthermore, although there is evidence that specific diet-derived microRNA (e.g., from rice) can be found in blood, more systematic and comprehensive characterization of blood exRNA is needed to determine the extent to which environmentally-derived RNAs are present in the human circulation. To characterize the spectrum of endogenously and exogenously-derived exRNAs present in human body fluids, we will analyze unique cohorts of well-defined specimens including: (i) well-characterized healthy cohorts of matched plasma, urine and serum from an observational epidemiology study with careful participant selection and well-controlled collection of samples and metadata, (ii) serial plasma samples collected before and after controlled feeding of vegetables in a study of healthy volunteers, and (iii) a cohort of well- characterized semen specimens from healthy men. We have assembled a team of experts to carry out this study including an experienced epidemiologist who has directly overseen collection of all of the plasma, urine and serum specimens, internationally-renowned expertise in the biology of extracellular RNAs in human body fluids, expertise in comprehensive quality assessment of specimens and quality-controlled purification of both extracellular vesicle-encapsulated and protein-bound forms of exRNA, and production-quality expertise in high- throughput sequencing and associated RNA bioinformatics and statistical analysis. We will perform quality assessment, fractionation of vesicle and non-vesicle-associated fractions, and high-throughput sequencing using multiple library preparation to capture as vast a space as possible of exRNAs in the banked human specimens mentioned above. We will use a bioinformatic pipeline to annotate both endogenous and potential exogenously-derived exRNAs, using the design of the controlled vegetable feeding study as a powerful tool to unambiguously assign exogenous origin to specific plant-derived RNAs. Finally, we will also use cutting-edge droplet digital PCR technology to validate and provide absolute quantification of exRNAs of interest, which is essential for establishing reference ranges describing variation in the healthy population.

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