The dose of PEG-IFNα-2b was reduced to 0.75 μg kg−1 week−1 when either neutrophil count was less than 750/mm3 or platelet count was less than 80 × 103/mm3. The dose of RBV was reduced to 600 mg/day when the hemoglobin concentration decreased to 10 g/dL. More than 80% adherence was achieved in all patients. Liver biopsy was performed immediately before initiating the therapy. After extraction of total RNA from liver biopsy specimens, the messenger RNA (mRNA) expression of the positive and negative cytoplasmic viral sensor (RIG-I, MDA5, Selleckchem RG7420 and LGP2), the adaptor molecule (IPS-1), the

related ubiquitin E3-ligase (RNF125), the modulators of these molecules (ISG15 and USP18), and IFNλ (IL28A/B) was quantified by real-time quantitative polymerase chain reaction (PCR) using target gene-specific primers. In brief, total RNA was

extracted by the acid-guanidinium-phenol-chloroform method using Isogen reagent (Nippon Gene, Toyama, Japan) from the liver biopsy specimen, which was 0.2-0.4 cm in length and 13G in diameter. Complementary DNA (cDNA) was transcribed from 2 μg of total RNA template in a 140-μL reaction mixture using the SYBR RT-PCR Kit (Takara Bio, Otsu, Japan) with random hexamer. Real-time quantitative PCR was performed using Smart Cycler version II (Takara Bio) with the SYBR RT-PCR Kit (Takara Bio) according to the manufacturer’s instructions. Assays were performed in duplicate selleckchem click here and the expression levels of target genes were normalized to the expressions of glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) gene and hydroxymethylbilane synthase (HMBS), an enzyme that is stable in the liver, as quantified using real-time quantitative PCR as internal controls. For accurate normalization, a set of two housekeeping genes was used in the present study. Sequences of the primer sets were as follows: RIG-I, 5′-AAAGCATGCA TGGTGTTCCAGA-3′, 5′-TCATTCGTGCATGCTC ACTGATAA-3′; MDA5, 5′-ACATAACAGCAACATG GGCAGTG-3′, 5′-TTTGGTAAGGCCTGAGCTGG AG-3′; LGP2, 5′-ACAGCCTTGCAAACAGTACAAC CTC-3′, 5′-GTCCCAAATTTCCGGCTCAAC-3′; IPS-1, 5′-GGTGCCATCCAAAGTGCCTACTA-3′, 5′-CAGC ACGCCAGGCTTACTCA-3′; RNF125, 5′-AGGGCA CATATTCGGACTTGTCA-3′, 5′-CGGGTATTAAAC GGCAAAGTGG-3′; ISG15, 5′-AGCGAACTCATCT TTGCCAGTACA-3′, 5′-CAGCTCTGACACCGACA TGGA-3′; USP18, 5′-TGGTTCTGCTTCAATGACT CCAATA-3′, 5′-TTTGGGCATTTCCATTAGCACT C-3′; IFNλ: 5′-CAGCTGCAGGTGAGGGA-3′, 5′-G GTGGCCTCCAGAACCTT-3′; GAPDH, 5′-GCACC GTCAAGGCTGAGAAC-3′, 5′-ATGGTGGTGAAGA CGCCAGT-3′; HMBS, 5′-AAGCGGAGCCATGTCT GGTAAC-3′, 5′-GTACCCACGCGAATCACTCTCA-3′. Genetic polymorphism in a tagged SNP located near the IL28B gene (rs8099917 and rs12979860) was determined by direct sequencing of PCR-amplified DNA. In brief, after extraction from whole blood samples, genomic DNA was amplified by PCR.