【系列2】胎牛血清RNA干擾了細胞培養外源性RNA

To further evaluate the potential of FBS RNA to interfere with cell culture derived exRNA, we performed RNA deep sequencing of the FBS fractions. Three different batches of FBS were spun at 100,000g for 24h, and the RNA samples isolated from the pelleted EV-enriched fractions and EV-depleted supernatants were used for the construction of the NEBNext small RNA libraries, followed by the HiSeq 2000 RNA-seq. Read mapping to the hg19 version of Human Genome was performed using the exceRpt small RNA-seq pipeline V2.2.8 of Genboree Workbench with default parameters14. On average, 13.6% and 21.7% reads from the “pellet RNA” and “supernatant RNA” fractions, respectively, were mapped to the human genome, suggesting that a significant part of FBS-associated transcripts is evolutionally conserved and could contribute to false-positives in the analysis of human cells. With precise no-mismatch-only mapping allowed, 9.2% and 13.2% reads of the respective fractions were mapped to the human genome. Major RNA composition of the two fractions, represented as reads per million mapped (RPM), is shown in Fig. 1d. Notably, both EV-enriched and EV-depleted fractions contained diverse RNA species, including mRNA, rRNA, miRNA, and other non-coding transcripts (antisense, snoRNA, Y RNA, etc.). The differences between the two sets of fractions were largely quantitative, with miRNA and rRNA fragments relatively enriched in the pelleted fractions, and mRNA and snoRNA – in the supernatants. Based on the RNA repertoire, Pearson clustering analysis demonstrated a clear separation between EV-enriched and EV-depleted fractions (Fig. 1e).

From 5.2% to 12.9% reads mapped to human genome corresponded to miRNA. MiR-122 was the most abundant miRNA in the FBS, followed by miR-1246, miR-423-5p, miR-148a-3p, and let-7 family (Fig. 1f). High levels of these miRNAs in the FBS were further confirmed by qRT-PCR (Fig. 1g). The depletion efficiencies, defined as abundance ratios between the pellet and supernatant fractions, were substantially different for these miRNAs, suggesting their distinct association with EVs and RNPs. Of note, all miRNAs mentioned above have been previously reported as enriched in cell culture-derived exRNA relative to cellular RNA8,10,15,16. It might be worth revisiting those results as they do not take FBS RNA into account and almost certainly overestimate secretion and enrichment of specific exRNA species in the conditioned media. FBS RNA reads were also mapped to mouse genome (Fig. 1f), indicating that FBS RNA could affect the results of both human and rodent culture exRNA analysis. Therefore, despite low levels of FBS RNA, its contribution should be carefully considered in the exRNA research, as misannotated bovine transcripts will lead to false positives and misinterpretations, especially when the determinants of RNA release and enrichment in extracellular complexes are investigated.