【系列2】胎牛血清RNA干擾了細胞培養外源性RNA

Several observations suggested that FBS-associated RNA might interfere with the analysis of exRNA. First, we detected high levels of miR-122, a specific miRNA abundantly expressed in liver but undetected in other tissues2,3, in the conditioned media of glioma cell lines cultured as a monolayer or neurospheres (Fig. 1a). Considering the lack of miR-122 expression in glioblastoma tumors4 and cultured glioma cells (Fig. 1a), we rationalized that this finding could be explained by either unusually efficient miR-122 secretion, or more plausibly, culture media as a primary source of this miRNA. Indeed, miR-122 was highly abundant in fresh unconditioned media utilized for glioma cell cultures (Fig. 1b), indicating that the majority of miR-122 in the conditioned medium comes from the medium components rather than cell secretion. Another specific microRNA, miR-451a, expressed in diverse cells and tissues with highest abundance in the blood2, was reported as the most enriched miRNA in EVs secreted by various cell types5,6,7,8,9,10,11. Consistent with these findings, we found miR-451a enriched in the pelleted EVs isolated from the conditioned media of U251 and 20/3 glioma monolayer cultures grown in DMEM/10% vdFBS (Fig. 1a). However, when the same cell lines were cultured at the same glucose concentration but in serum-free neurosphere-promoting conditions, miR-451a was not detected in the ultracentrifugation (UC) pellets/exRNA fraction (Fig. 1a). To better understand the contribution of different culture media, we isolated total exRNAs from fresh unconditioned “monolayer culture medium” (2D medium, containing DMEM/10% FBS), vesicle-depleted 2D medium (vd2D, containing FBS after 24h UC), and “neurosphere medium” (3D medium, serum-free), and measured miR-451a levels using qRT-PCR. MiR-451a was abundant in the 2D medium, slightly reduced in the vd2D medium, and barely detected in the 3D medium (Fig. 1b). Of note, the levels of miR-122 and miR-451 in the vd2D media relative to the crude 2D media were reduced to a different extent, but not entirely abolished. Although the possibility that culture conditions affect RNA secretion cannot be ruled out, these findings suggest that UC-based vesicle depletion does not fully eliminate RNA from the FBS/culture media, and that FBS-associated RNA may lead to false results and interpretation such as the previously reported miR-451a enrichment in exRNA.

(a) qRT-PCR analysis of 20/3 and U251 glioma cellular and EV-associated exRNA demonstrates that miR-122 and miR-451a are highly enriched in EVs when the cells are cultured with FBS (2D media). miR-451a is not detected in exRNA of cells cultured in the serum-free 3D medium. N=3 cells per group.

(b) miR-122 and miR-451a levels were determined in fresh media, including 2D medium containing 10% FBS, vd2D medium containing 10% FBS pre-spun for 24h, and serum-free 3D medium. Low Cq values correspond to high levels of the miRNA. N=3 media aliquots.