Ten nanograms of total RNA was reverse transcribed using the TaqMan microRNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) and microRNA-specific stem-loop primers (part of the TaqMan microRNA Assay Kit; Applied Biosystems). The above mixture was incubated at 37 °C for 60 min, 94 °C for 5 min and 4 °C for 10 min. Among the numerous target miRNAs, we examined 10 miRNAs that were increased or decreased in HCC tissue in previous studies.15 (link), 16 (link), 17 (link), 18 (link), 19 (link), 20 (link), 21 (link), 22 (link), 23 (link), 24 (link) To measure serum exosomal microRNAs, miR-18a, -21, -93, -106b, -221, -222 and -224 were selected as upregulated microRNAs, and miR-101, 122 and 195 were selected as downregulated microRNAs. The measurement of serum circulating microRNAs was performed for six microRNAs (miR-21, -221, -222, -224, -101 and -195). The real-time quantitative PCR for microRNAs was conducted according to the TaqMan miRNA assay protocol (Applied BioSystems). First, 1.5 μl RT product was combined with the TaqMan Universal PCR master mix and 0.5 μl probe mix of the TaqMan MicroRNA Assay. The total volume of the mixture product for PCR was 10 μl. The incubation of the mixture product was carried out at 95 °C for 10 min, followed by 40 cycles of 94 °C for 15 s and 60 °C for 1 min. Real-time quantitative PCR was performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems), and the results were analyzed using the RQ manager software (Applied Biosystems). The amplification plot reflecting the fluorescent signal at each cycle was determined based on the threshold cycle (Ct) values for each sample. The average of the Ct value was calculated after the PCRs were run in duplicate for each sample. The Cel-miR-39 value from the duplicate was used as the internal control.13 (link), 14 (link) The relative gene expression values for the target microRNA were normalized to Cel-miR-39 and calculated using the 2−ΔΔCT method.25 (link), 26 (link)