RNA Extraction and Gene Expression Analysis

10–20 mg of liver tissue was homogenized in QIAzol lysis reagent (QIAGEN, Valencia, CA, USA) using stainless steel beads via TissueLyser II (QIAGEN, Valencia, CA, USA). Total RNA was extracted using the miRNeasy kit (QIAGEN, Valencia, CA, USA) as recommended by the manufacturer.⁶⁰ (link) For mRNA analysis, cDNA was transcribed with the iScript reverse transcription system kit (Bio-Rad, Hercules, CA, USA), and quantitative real-time PCR was performed via CFX96 iCycler (Bio-Rad, Hercules, CA, USA). Quantitative analyses of genes were performed using gene-specific primers as presented in Table S2 and as described previously.⁶⁵ (link) Cq value was normalized to 18S or β actin mRNA, and differential expression fold changes were calculated using the delta-delta Ct method. For miR analysis, TaqMan miR assays (Applied Biosystems, Foster City, CA, USA) were used as described earlier.¹⁴ (link)^,60 (link) SnoRNA-202 (mouse samples) or RNU48 (human samples) were used to normalize the technical variations between the samples.