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Size-exclusion chromatography separation reveals that vesicular and non-vesicular small RNA profiles differ in cell free urine

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journal contribution
posted on 2021-06-25, 04:30 authored by J Karttunen, Sarah StewartSarah Stewart, L Kalmar, AJ Grant, FE Karet Frankl, TL Williams
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.


This work was funded by the PetPlan Charitable Trust, Grant number S18-600-638; Finnish Foundation of Veterinary Research (personal grant for Jenni Karttunen); the Academy of Medical Sciences Starter Grant for Clinical Lecturers (Tim L. Williams, Grant number SGL016 n1016); and a Biotechnology and Biological Sciences Research Council Future Leader Fellowship (Sarah Stewart, Grant number BB/P010911/1).


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International Journal of Molecular Sciences





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