Size-Exclusion Chromatography Separation Reveals That Vesicular and Non-Vesicular Small RNA Profiles Differ in Cell Free Urine |
| |
| Authors: | Jenni Karttunen Sarah E Stewart Lajos Kalmar Andrew J Grant Fiona E Karet Frankl Tim L Williams |
| |
| Affiliation: | 1.Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, UK; (J.K.); (L.K.); (A.J.G.);2.Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, University of Cambridge, Cambridge CB2 0QQ, UK;3.Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC 3086, Australia;4.Department of Medical Genetics, University of Cambridge, Cambridge CB2 0QQ, UK; |
| |
| Abstract: | Urinary 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. |
| |
| Keywords: | urine extracellular vesicles small RNA |
|
|
