Size-Exclusion Chromatography Separation Reveals That Vesicular and Non-Vesicular Small RNA Profiles Differ in Cell Free Urine

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.

Towards optimised extracellular vesicle proteomics from cerebrospinal fluid

“…In our study, NTA measurement showed a clear particle peak at SEC fraction 3, which is consistent with findings when EVs are isolated from other body fluids using the same method (Karttunen et al, 2021). In fraction 3, particles detected by NTA had a modal size of around 100nm, whereas particles measured by NanoFCM had a modal size of around 70nm.…”

“…The selection of isolation method is a balance between EV yield and purity, which poses challenges for EV biomarker discovery; optimising purity is likely to enhance test specificity, whilst optimising EV yield is necessary to maximise test sensitivity. Ultrafiltration combined with size-exclusion chromatography (UF-SEC) outperforms the differential centrifugation method and/or commercial precipitation-based kits for EV isolation from cell culture supernatant, and urine in terms of purity (Allelein et al, 2021; Karttunen et al, 2021; Sidhom et al, 2020). For CSF EVs, UF-SEC has been reported to increase both yield and purity (Thompson et al, 2018).…”

Exploration of Extracellular Vesicle miRNAs, Targeted mRNAs and Pathways in Prostate Cancer: Relation to Disease Status and Progression

“…Our study targeted miRNAs and mRNAs that were detected in small uEV-enriched samples isolated by ultracentrifugation, or pEV samples isolated with ExoEasy without further steps of purification or enzymatic treatments (RNAse/proteinase). These steps attempt to remove miRNA in other carriers than EV or miRNA associated with the molecular corona on the EV surface [23,27,74,75]. Our detected miRNAs can therefore be located in or on EV or in other small-sized miRNA carriers remaining in the samples, including recently identified supermeres [27,76].…”

“…However, so far, the heterogeneity of the uEV pipelines, on top of the heterogeneity of the PCa tumours and study designs, likely explains why the discovered miRNA biomarker candidates differ [8]. For example, heterogeneity of the isolated uEV and copurified non-EV materials from different isolation workflows impact the miRNA results [7,23,24]. As the average sizes, biogenesis routes and cargo of small and large EV differ [22,[25][26][27], targeting small EV for biomarker analysis is one strategy to limit heterogeneity of the EV and dissect exosomal messages.…”

Urinary extracellular vesicles as a source of protein‐based biomarkers in feline chronic kidney disease and hypertension

“…Briefly, buffer was removed from the columns which were then washed with PBS before instilling the resuspended precipitate or ultrafiltered urine, which was then followed by elution with PBS. The first 1000-μL fraction was discarded, and the following 600 μL collected as the EV rich fraction, as previously optimised (Karttunen et al 2021). Final uEV preparations were stored at −80°C until further analysis.…”