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Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000<jats:italic>g<\/jats:italic> to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.<\/jats:p>","DOI":"10.1038\/s41598-024-62783-9","type":"journal-article","created":{"date-parts":[[2024,5,28]],"date-time":"2024-05-28T22:02:03Z","timestamp":1716933723000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":23,"title":["Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation"],"prefix":"10.1038","volume":"14","author":[{"given":"Ana","family":"Teixeira-Marques","sequence":"first","affiliation":[]},{"given":"Sara","family":"Monteiro-Reis","sequence":"additional","affiliation":[]},{"given":"Diana","family":"Montezuma","sequence":"additional","affiliation":[]},{"given":"Catarina","family":"Louren\u00e7o","sequence":"additional","affiliation":[]},{"given":"Miguel Carlos","family":"Oliveira","sequence":"additional","affiliation":[]},{"given":"Vera","family":"Const\u00e2ncio","sequence":"additional","affiliation":[]},{"given":"Jos\u00e9 Pedro","family":"Sequeira","sequence":"additional","affiliation":[]},{"given":"Carina","family":"Carvalho-Maia","sequence":"additional","affiliation":[]},{"given":"Rui","family":"Freitas","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5363-2525","authenticated-orcid":false,"given":"Elena S.","family":"Martens-Uzunova","sequence":"additional","affiliation":[]},{"given":"M. Helena","family":"Vasconcelos","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3171-4666","authenticated-orcid":false,"given":"Rui","family":"Henrique","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4186-5345","authenticated-orcid":false,"given":"Carmen","family":"Jer\u00f3nimo","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2024,5,28]]},"reference":[{"key":"62783_CR1","doi-asserted-by":"publisher","unstructured":"Oeyen, E. et al. Bladder cancer diagnosis and follow-up: The current status and possible role of extracellular vesicles. Int. J. Mol. Sci. 20. https:\/\/doi.org\/10.3390\/ijms20040821 (2019).","DOI":"10.3390\/ijms20040821"},{"key":"62783_CR2","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1126\/science.aau6977","volume":"367","author":"R Kalluri","year":"2020","unstructured":"Kalluri, R. & LeBleu, V. S. 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