RNA was isolated using RNeasy FFPE Mini Kit (QIAGEN, selleck chemical Hilden, Germany) according to
the manufacturer’s instructions with modification for copurification of miRNA, then stored at −80°C. Contaminating genomic DNA was removed using Turbo DNase digestion (Ambion Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. MicroRNA expression was determined applying the following TaqMan MicroRNA Assays (Applied Biosystems, Carlsbad, CA, USA): miR-21 (ID:000397), miR-23a (ID:000399), miR-34a (ID:000426), miR-96 (ID:000186), miR-99a* (ID:002141), miR-122 (ID:002245), miR-125b (ID:000449), miR-181a-2* (ID:002317), miR-194 (ID:000493), miR-195 (ID:000494), miR-217 (ID:002337), miR-221 (ID000524), and miR-224 (ID:002099). RT
was performed using TaqMan Ulixertinib manufacturer MicroRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions in 7.5 μL final volume containing 10 ng total RNA. TaqMan Universal PCR Master Mix No AmpErase UNG (Applied Biosystems) was applied for real-time PCR, also carried out according to the manufacturer’s instructions, in 10 μL final volume containing 0.65 μL RT product. The reaction was run on an ABI PRISM 7000 system (Applied Biosystems). The samples were measured in duplicates. Relative expression level in samples was determined by 2ΔCq method using the mean Cq value of miR-23a and miR-34a as reference. These 上海皓元 reference miRs showing the overall least variation among samples were selected using NormFinder[20] as U6 snRNA (ID:001973) proved to be highly variable in the samples. SPSS 15. version (SPSS, Inc., Chicago, IL, USA) was used for the statistical analysis. Continuous variables
are shown as mean values and standard deviations. Student’s t-test, anova test with Scheefe and Bonferroni post hoc tests, as well as Mann–Whitney U-test were utilized for univariate analyses, after examining population homogeneity of the variables (Levene test). The anova method was used to compare microRNA expression before and after IFN therapy, when therapy response (SVR vs NR) was taken into account. The connections between continuous variables were evaluated by correlation analysis, using Pearson correlation coefficient. P < 0.05 was considered significant. Baseline characteristics of patients are shown in Table 1. Examination of the clinical data revealed that post-treatment viral load decreased significantly only in the SVR group when compared with pretreatment levels (12 × 106 /mL ± 15.7 vs 0/mL, P = 0.003). HAI decreased in both NR (3.74 ± 1 vs 1.6 ± 1, P < 0.0001) and SVR groups (4 ± 1 vs 1 ± 1.3, P = 0.002) after administering IFN/RBV therapy. After finishing antiviral therapy fibrosis score was analyzed, and no correlation was found between fibrosis being above or below the median (fibrosis score 1) and the investigated miRs. High viral load (above median 3.