Abstract
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, we established an experimental pipeline for comprehensive profiling of small exRNAs isolated from cell culture. By evaluating six RNA extraction protocols, we developed an improved method for robust recovery of vesicle-bound exRNAs. With this method, we 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, we 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, we 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, our work has laid a solid foundation for further standardization on exRNA analysis across various cellular systems.
Highlights
Extracellular vesicles (EVs) are lipid-bilayer particles that are released from many types of c ells[1,2]
EXOs and MVs were both highly enriched in tRNAs and ribosomal RNA (rRNA), but depleted in small nucleolar RNA (snoRNA)
Similar observations on tRNA composition of Extracellular RNAs (exRNAs) were reported previously based on a limited number of cell lines[1,24,25]
Summary
Extracellular vesicles (EVs) are lipid-bilayer particles that are released from many types of c ells[1,2]. It is important to establish a robust standardized exRNA extraction method that can be broadly applied to most cell biology studies[18,19]. To address this challenge, we developed an improved pipeline for exRNA isolation and small RNA-seq profiling analysis. We developed an improved pipeline for exRNA isolation and small RNA-seq profiling analysis Using this pipeline, we performed small RNA-seq for EXOs, MVs and source cells from 14 cancer cell lines. We performed small RNA-seq for EXOs, MVs and source cells from 14 cancer cell lines In this way, we were able to comprehensively identify common as well as cell specific exRNA expression profiles in human papillomavirus (HPV)-induced cancers
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