【系列2】胎牛血清RNA干擾了細胞培養外源性RNA

(c) Percent of total RNA isolated from FBS pelleted and supernatant fractions after UC for 80min, 5hr, or 24hr. N=3 FBS aliquots per group with two-tailed t-test. (d) RNA-seq analysis shows RNA composition of FBS pellets or supernatants (24hr UC). The reads were mapped to either human or mouse genomes, and the data is shown as mean?±?SEM RPM. N=3 FBS aliquots. (e) Pearson clustering analysis of logarithm-transformed data based on major RNA composition shows separation of three supernatant samples (denoted as S) from three pellet samples (denoted as P). (f) The most abundant miRNAs in FBS pellet and supernatant fractions (24h UC), as detected by RNA-seq. Of note, miR-451 was not sequenced from the NEBNext small RNA libraries. (g) The levels of selected miRNAs in FBS pellets and supernatants were determined by qRT-PCR. N=3 FBS aliquots per group. (h) Bovine and primate-specific miR-1246 is detected by qRT-PCR in mouse cultured cells but not mouse tissues. N=3 cells or tissue slices per group. *p<0.05; **p<0.01; NS, not significant. All bars represent mean?±?SEM in the figure.

RNA cannot be depleted from FBS using ultracentrifugation

We next set to characterize the composition of FBS RNA and investigate whether RNA can be removed from serum. Serum RNA is associated with exosomes and other EVs, as well as non-membrane lipid and protein RNPs. Most researchers use 70,000g to 110,000g UC for 2h–18h to produce vdFBS. It has been demonstrated that prolonged UC removes the majority, although not all EVs, as monitored by NanoSight12. However, to what extend the vdFBS is RNA-depleted is unclear. To address this question, we spun FBS samples at 100,000g for 80min, 5h, or 24h and isolated total RNA from the pelleted (i.e. EV-enriched) material and the supernatants (i.e. EV-depleted). RNA was also isolated from crude FBS at the baseline. 5–40ng/ml total RNA was isolated from the crude FBS, varying between the scales and batches, and consistent with the previous assessments of human serum13. As shown in Fig. 1c, even the prolonged 24-hour UC extended beyond the reported protocols, removed only a part of the RNA (19–33%) from FBS (Fig. 1c).On average, the vdFBS preparations contained 34ng/ml RNA, the majority of which was most likely associated with non-vesicular complexes, such as RNPs. Regardless of the source of this RNA (vesicular or not), it can potentially contaminate cell culture condition media and co-isolate/precipitate with the cell culture EVs in the subsequent procedures.

FBS contains a diverse repertoire of conserved RNA species indistinguishable from human and mouse transcripts