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dc.contributor.authorKarttunen, Jenni
dc.contributor.authorStewart, Sarah E
dc.contributor.authorKalmar, Lajos
dc.contributor.authorGrant, Andrew J
dc.contributor.authorKaret Frankl, Fiona E
dc.contributor.authorWilliams, Tim L
dc.date.accessioned2021-07-07T13:59:32Z
dc.date.available2021-07-07T13:59:32Z
dc.date.issued2021-05-05
dc.identifier.issn1422-0067
dc.identifier.otherPMC8124894
dc.identifier.other34063036
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/324849
dc.description.abstractUrinary extracellular vesicles (EVs) and their RNA cargo are a novel source of biomarkers for various diseases. We aimed to identify the optimal method for isolating small (<200 nm) EVs from human urine prior to small RNA analysis. EVs from filtered healthy volunteer urine were concentrated using three methods: ultracentrifugation (UC); a precipitation-based kit (PR); and ultrafiltration (UF). EVs were further purified by size-exclusion chromatography (SEC). EV preparations were analysed with transmission electron microscopy (TEM), Western blotting, nanoparticle tracking analysis (NTA) and an Agilent Bioanalyzer Small RNA kit. UF yielded the highest number of particles both before and after SEC. Small RNA analysis from UF-concentrated urine identified two major peaks at 10-40 nucleotides (nt) and 40-80 nt. In contrast, EV preparations obtained after UC, PR or SEC combined with any concentrating method, contained predominantly 40-80 nt sized small RNA. Protein fractions from UF+SEC contained small RNA of 10-40 nt in size (consistent with miRNAs). These data indicate that most of the microRNA-sized RNAs in filtered urine are not associated with small-sized EVs, and highlights the importance of removing non-vesicular proteins and RNA from urine EV preparations prior to small RNA analysis.
dc.languageeng
dc.rightsAttribution 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.sourceessn: 1422-0067
dc.sourcenlmid: 101092791
dc.subjectUrine
dc.subjectSmall RNA
dc.subjectExtracellular Vesicles
dc.subjectCell-Free System
dc.subjectHumans
dc.subjectMicroRNAs
dc.subjectUltrafiltration
dc.subjectUltracentrifugation
dc.subjectChromatography, Gel
dc.titleSize-Exclusion Chromatography Separation Reveals That Vesicular and Non-Vesicular Small RNA Profiles Differ in Cell Free Urine.
dc.typeArticle
dc.date.updated2021-07-07T13:59:31Z
prism.issueIdentifier9
prism.publicationNameInternational journal of molecular sciences
prism.volume22
dc.identifier.doi10.17863/CAM.72303
rioxxterms.versionofrecord10.3390/ijms22094881
rioxxterms.versionVoR
rioxxterms.licenseref.urihttps://creativecommons.org/licenses/by/4.0/
dc.contributor.orcidKarttunen, Jenni [0000-0002-0099-1305]
dc.contributor.orcidStewart, Sarah E [0000-0003-3712-9898]
dc.contributor.orcidWilliams, Tim L [0000-0002-9775-8285]
pubs.funder-project-idBiotechnology and Biological Sciences Research Council (BB/P010911/1)
pubs.funder-project-idPetplan Charitable Trust (S18-600-638)
pubs.funder-project-idAcademy of Medical Sciences Starter Grant for Clinical Lecturers (SGL016\1016)
pubs.funder-project-idSuomen Eläinlääketieteen Säätiö (personal grant for Jenni Karttunen)


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International