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Serum-free media supplements carry miRNAs that co-purify with extracellular vesicles
Ist Teil von
Journal of extracellular vesicles, 2018-01, Vol.7, p.150-150
Ort / Verlag
Abingdon: John Wiley & Sons, Inc
Erscheinungsjahr
2018
Link zum Volltext
Quelle
Wiley Online Library Journals Frontfile Complete
Beschreibungen/Notizen
Background: Numerous studies report the association of miRNAs with extracellular vesicles (EVs). In most cases, EVs were harvested from cell culture-conditioned media containing serum or a defined media supplement as nutrient. We are analysing miRNAs associated with EVs derived from oligodendrocytes, which deliver EV-associated cargo to neurons. Since a recent study revealed miRNAs in vesicle-depleted-foetal bovine serum medium co-isolating with EVs, we controlled media supplements routinely used for neural cell culture for the presence of miRNAs that had been identified in a RNA-Seq data set of oligodendrocytederived EVs. Methods: We characterized the topology of RNAs associated with oligodendroglial EVs by enzymatic digests. We furthermore performed RNA-Seq of small RNAs purified from EVs isolated from primary cultured oligodendrocytes by differential centrifugation to reveal EVenriched miRNAs. Validation of miRNAs was performed by qPCR of miRNAs isolated from purified EVs as well as un-conditioned media or the media supplements NB21 and B27 subjected to the EV-isolation protocol. To reveal potential sources of miRNAs, individual media components were assessed by qPCR. Results: Enzymatic digestion of isolated EVs using RNAse and protease indicated that oligodendroglia-derived EVs contain a distinct population of small RNAs. Intriguingly, validation of miRNAs identified by RNASeq revealed that most EV-associated miRNAs were robustly detected in un-conditioned media and the media supplements NB21 and B27. By screening individual supplement components, we were able to exclude bovine serum albumin as major source of miRNA contamination and identified a single component as carrier of miRNAs. Summary/Conclusion: Our study shows that a single component of defined media supplements may carry major contaminating miRNAs into EV samples. Hence, EV RNA-Seq data should be carefully controlled. Our study identifies the major contaminating source and may help to formulate miRNA-free media supplements in the future.