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Although interest in EV translation is ever\u2010increasing, development efforts are hampered by the inability to reliably assess the uptake of EVs and their RNA cargo. Here, we establish a novel qPCR\u2010based method for the detection of unmodified EVs using an RNA Tracer (DUST). In this proof\u2010of\u2010concept study we use a human\u2010specific Y RNA\u2010derived small RNA (YsRNA) we dub \u201cNT4\u201d that is enriched in cardiosphere\u2010derived cell small EVs (CDC\u2010sEVs). The assay is robust, sensitive, and reproducible. Intravenously administered CDC\u2010sEVs accumulated primarily in the heart on a per mg basis. Cardiac injury enhanced EV uptake in the heart, liver, and brain. Inhibition of EV docking by heparin suppressed uptake variably, while inhibition of endocytosis attenuated uptake in all organs. In vitro, EVs were uptaken more efficiently by macrophages, endothelial cells, and cardiac fibroblasts compared to cardiomyocytes. These findings demonstrate the utility of DUST to assess uptake of EVs in vivo and in vitro.<\/jats:p>","DOI":"10.1002\/jev2.12178","type":"journal-article","created":{"date-parts":[[2022,1,10]],"date-time":"2022-01-10T04:55:16Z","timestamp":1641790516000},"source":"Crossref","is-referenced-by-count":30,"title":["Biodistribution of unmodified cardiosphere\u2010derived cell extracellular vesicles using single RNA tracing"],"prefix":"10.1002","volume":"11","author":[{"given":"Alessandra","family":"Ciullo","sequence":"first","affiliation":[{"name":"Smidt Heart Institute Cedars\u2010Sinai Medical Center  Los Angeles California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chang","family":"Li","sequence":"additional","affiliation":[{"name":"Smidt Heart Institute Cedars\u2010Sinai Medical Center  Los Angeles California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liang","family":"Li","sequence":"additional","affiliation":[{"name":"Smidt Heart 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