All primer and probes of each miRNA investigated were present in the TaqMan microRNA assays purchased from Applied Biosystems. MiRNAs were extracted from the cells using the mirVana miRNA Isolation Kit (Ambion, Austin, TX), according to the manufacturer’s protocol. Applied Biosystems TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Monza, Italy) was used (following the despite manufacturer’s protocol) for reverse transcription (RT) of extracted total miRNAs. Each RT reaction contained 5 ng of extracted total miRNA, 3 ��L of TaqMan MicroRNA assays, 1.50 ��L of RT10x buffer, 0.25 mM each of dNTPs, 3.33 U/��L Multiscribe reverse transcriptase and 0.25 U/��L RNase inhibitor. The 15 ��L reactions were incubated in a Biometra T3 Thermocycler (MMedical, Italy) in a 96-well plate for 30 minutes at 16��C, 30 minutes at 42��C, 5 minutes at 85��C, and then held at 4��C.

For the real-time PCR step, amplification was carried out using TaqMan MicroRNA assays (Applied Biosystems) on the Applied Biosystems 7000 Real-Time PCR system. The 20 ��L reaction included 1.33 ��L RT product, 10 ��L of TaqMan Universal PCR Master Mix with no UNG and 1 ��L of TaqMan MicroRNA assays. The reactions were incubated in a 96-well optical plate at 95��C for 10 minutes, following by 40 cycles of 95��C for 15 s and 60��C for 1 minute. Real-time PCRs for each miRNA were run in triplicate. The relative expression levels of each miRNA were measured using the constitutively expressed RNU6B as endogenous control The expression of each miRNA relative to RNU6B was determined using the arithmetic formula (2 – ��Ct) or (2 – ����Ct) according to the supplier’s guidelines (Applied Biosystems).

Statistical analysis All results are expressed as the mean �� standard deviation (median). The coefficient of variation (CV) was used to measure the interpatient variability in blood concentrations of miRNAs and MxA. Levels of miRNAs and MxA observed in PBMCs from three healthy individuals Drug_discovery before and after the stimulation in vitro with IFN alpha were compared using a T-test as suggested by Bland and Altman [16]. Differences between patients with CHC and healthy individuals, and between patient groups, in terms of blood concentrations in miRNAs and MxA, were compared using the Wilcoxon test. The same test was used to assess differences between miRNAs and MxA expression levels in patients with CHC. A Spearmen rho coefficient was calculated to assess the correlation between pre-dose and HCV viral load, ALT status. Significance was fixed at the 5% level. Analysis was performed using spss version 13.0 for Windows.