Extracellular RNAs (exRNAs) have attracted great attention due to their essential role in cell-to-cell communication as well as their potential as non-invasive disease biomarkers. However, at present, there is no consensus on the best method to profile exRNA expression, which leads to significant variability across studies. To address this issue, researchers at the Washington University School of Medicine established an experimental pipeline for comprehensive profiling of small exRNAs isolated from cell culture. By evaluating six RNA extraction protocols, they developed an improved method for robust recovery of vesicle-bound exRNAs. With this method, the researchers performed small RNA sequencing of exosomes (EXOs), microvesicles (MVs) and source cells from 14 cancer cell lines. Compared to cells, EXOs and MVs were similarly enriched in tRNAs and rRNAs, but depleted in snoRNAs. By miRNA profiling analysis, they identified a subset of miRNAs, most noticeably miR-122-5p, that were significantly over-represented in EXOs and MVs across all 14 cell lines. In addition, they also identified a subset of EXO miRNAs associated with cancer type or human papillomavirus (HPV) status, suggesting their potential roles in HPV-induced cancers. In summary, this work has laid a solid foundation for further standardization on exRNA analysis across various cellular systems.
The small RNA composition of EXOs, MVs and cells
(A) The small RNA composition in all samples combined. (B) The tRNA composition of EXOs and MVs. (C) The percentage of three most abundant tRNA species in EXOs, MVs and cells.