Study design for circulating microRNA expression analysis in patients with chronic hepatitis C. We enrolled 130 CHC patients from the anti-HCV antibody positive blood donor cohort. Initially, we analyzed miRNA expression patterns in plasma in the discovery set consisting of 16 healthy controls, 16 patients with mild fibrosis and 16 with significant fibrosis. Then, we verified the microarray results by qRT-PCR using the same samples, and selected candidate miRNAs for further study. Next, we analyzed the association between the candidate miRNA levels in plasma or EVs and clinical features in the entire set that included the 32 patients in the discovery set. Finally, we analyzed changes in the candidate miRNA levels in plasma between the dates of paired liver biopsies in the longitudinal set. HCV, hepatitis C virus; miRNA, microRNA; CHC, chronic hepatitis C; HC, healthy control; qRT-PCR, quantitative real-time polymerase chain reaction; EVs, extracellular vesicles.