Impact of heroin use and/or HCV infection on HCV-related circulating MicroRNAs. Plasma specimens were collected from control subjects (n = 20), heroin users with (n = 20) or without (n = 20) HCV infection, and subjects with HCV infection but without heroin use (n= 20), respectively. Total RNA was extracted from plasma and subjected to real-time RT-PCR to quantify circulating miRNA-122 (a), -141 (b), -351 (c), -29a (d), -29b (e), and -29c (f). Synthetic C. elegans miRNA-39 (celmiR-39) was used as a spiked-in miRNA. Levels of target circulating miRNAs were expressed by normalization to the spiked-in cel-miR-39 and compared between the 4 groups. Data are shown as scatter plots. Mean values are indicated by horizontal bars. Statistical significance was calculated by the one-way ANOVA with Tukey-Kramer adjustment for multiple comparisons