RNA extracted from human liver tissue was used for quantification of STAT1, IP10, USP18, IFI27, Viperin and IFI44L messenger RNAs (mRNAs). Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Basel, Switzerland) according to manufacturer’s instructions. RNA was aliquoted and stored at −75°C. RNA was reverse transcribed by Moloney murine leukemia virus reverse transcriptase (Promega Biosciences, Wallisellen, Switzerland) in the presence of random hexamers (Promega) and deoxynucleoside triphosphates. The SYBR-PCR reactions were performed using the SYBR green PCR
master mix (Applied Biosystems) and primers spanning the exon-intron junctions to avoid amplification of genomic DNA. The following primers were used: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GCTCCTCCTGTTCGACAGTCA-3′ and 5′-ACCTTCCCCATGGTGT- find more CTGA-3′; STAT1, 5′-TCCCCAGGCCCTTGTTG-3′ and 5′-CAAGCTGCTGAAGTTGGTACCA-3′; IP10, 5′-CGATTCTGATTTGCTGCCTTAT-3′ and 5′-GCAGGTACAGCGTACGGTTCT-3′; USP18, 5′-CTCAGT- CCCGACGTGGAACT-3′ and 5′-ATCTCTCAAGCGC- Temozolomide clinical trial CATGCA-3′; IFI27, 5′-CCTCGGGCAGCCTTGTG-3′ and 5′-AATCCGGAGAGTCCAGTTGCT-3′; Viperin, 5′-CTTTGTGCTGCCCCTTGAG-3′ and 5′-TCCATACCAGCTTCCTTAAGCAA-3′; IFI44L, 5′-GCTGCGGGCTGCAGAT-3′ and 5′-CTCTCTCAATTGCACC- AGTTTCC-3′. The difference in the cycle threshold (ΔCt) value was derived by subtracting the Ct value for GAPDH, which
served as internal control, from the Ct value for transcripts of interest. All reactions were run in duplicate,
using an Applied Biosystems Prism 7000 Sequence Detection System. Messenger RNA expression levels were calculated relative to GAPDH from the ΔCt values, using the formula 2−ΔCt. One microgram total RNA isolated from biopsy specimens of 44 CHC patients was reverse transcribed according to the manufacturer’s instructions using a pathogen-specific RT primer mix (PrimerDesign, Southampton, UK) designed for the in vitro quantification of all HCV genotypes. HCV RNA was quantified using a pathogen-specific primer/probe mix (PrimerDesign) and the Taqman Universal PCR Master Mix (Applied Biosystems, manufactured by Roche, NJ). Fluorescence was detected through the FAM channel of the Applied Biosystems 7000 Sequence Detection System, and copy number of HCV RNA per microgram total RNA PTK6 was calculated according to the standard curve obtained using control template (PrimerDesign). Correlations were assessed using the Spearman coefficient. Comparisons between two groups or between multiple groups were performed with the Mann-Whitney test and one-way analysis of variance, respectively, using GraphPad Prism version 4.00 for Macintosh (GraphPad Software, San Diego, CA). A P ≤ 0.05 was considered as statistically significant. The clinical characteristics of all CHC patients and controls who were part of this study are summarized in Table 1.