MicroRNA expression profiling of tumor cells derived exosomes

In this study, we isolated exosomes from liver cancer cell lines and exosomal miRNA expression was profiled by high-throughput sequencing to investigate the potential of exosomal contents as biomarkers for HCC. Overall design: 97H or HepG2 cells were cultured using DMEM medium containing 10% exosom-free serum. The exosomes were extracted from the culture supernatant by ultra-high speed centrifugation. Exosomes were identified and verified using transmission electron microscopy, nano-flow cytometry analysis, and western blotting. Total RNA, including miRNA, was isolated from exosome using Trizol reagent. RNA was applied for small RNA library construction using the NEB Multiplex Small RNA Library Prep Set for Illumina. The purified library was examined using the Agilent 2100 Bioanalyzer and the Agilent DNA 1000 chip kit for quality control and then sequenced using the Illumina NextSeq 500 platform.