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.

Public Health Relevance

In this project, we will discover the composition and sequence of RNA molecules present outside of cells in the blood, urine and semen fluid of healthy people. This knowledge is needed to for understanding how RNA molecules may function as hormones in the body, for developing new blood tests to diagnose disease using RNA molecules, and for using RNA molecules present outside of cells to develop therapies for a variety of diseases.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project--Cooperative Agreements (U01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-GGG-A (51))
Program Officer
Srinivas, Pothur R
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Michigan Ann Arbor
Internal Medicine/Medicine
Schools of Medicine
Ann Arbor
United States
Zip Code
Giraldez, Maria D; Spengler, Ryan M; Etheridge, Alton et al. (2018) Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling. Nat Biotechnol 36:746-757
Grossman, R L; Abel, B; Angiuoli, S et al. (2017) Collaborating to Compete: Blood Profiling Atlas in Cancer (BloodPAC) Consortium. Clin Pharmacol Ther 101:589-592
Tewari, Muneesh (2015) A functional extracellular transcriptome in animals? Implications for biology, disease and medicine. Genome Biol 16:47