Small RNA sequencing of FBS and FBS fractions (extracellular vesicles and RNA-associated proteins)

Exosomes are extracellular vesicles that range from 30 to 100 nm approximately and can mediate cargo transfer into a wide variety of recipient cells. Due to this, exosomes have emerged as a strong vehicle for small molecules and drug delivery.Recently, it was reported that microRNAs (miRNAs) contained extracellular vesicles (EVs) from Fetal Bovine Serum (FBS) could be transferred into cell cultures. Wei et al. separated vesicular from non-vesicular fractions by ultracentrifugation and concluded that the fraction containing EVs could mediate miRNAs transfer into cell cultures, potentially biasing the results of small RNA detection; RNA classes from FBS were detected by deep sequencing after FBS fractionation by ultracentrifugation. However, it is widely known that exosome pellets resulting from ultracentrifugation are often contaminated with non-vesicular proteins (such as argonaute), leaving open the possibility that the identity for some RNAs, or their abundance, may be misrepresented. Here, we present a procedure to obtain highly purified fraction of EVs from FBS using size exclusion chromatography followed by pelletization via Vn96 peptide, in order to characterize the RNAs present in exosomes, as compared with the RNAs from non-vesicular fractions from FBS. Our results showed that this method is easily scalable and removes a large amount of serum albumin, the most abundant protein component of FBS. We identified RNA classes contained in six different brands of FBS and also characterized the fraction containing EVs, and the non-EVs fraction separately (protein-associated RNA). By using our two-step purification we found that there are sets of RNAs that are mutually exclusive between fractions and yet another group that it is, apparently, shared between them. The differences between the EVs and whole FBS classes of RNA might be important when similar results are found in others biofluids that are used —or proposed as biomarkers, due to some RNAs present in the non-vesicular fraction could potentially interfere with diagnosis.