Liver biopsy RNA Extraction and Microarray Analysis

Core liver biopsies were immediately placed in RNA later (Qiagen) and stored at −80°C until handling. Total RNA extraction from liver tissue was performed on paired liver biopsies using RNeasy kit (Qiagen). The quality of isolated total RNA was assessed using bioanalyzer, (Agilent Technologies, CA) and RIN (RNA Integrity Number) values were generally above 8.0 for all but three samples. Ten nanograms of total RNA from each sample was used for cDNA library preparation using Ovation Pico WTA system V2 kit (NuGEN Technologies Inc, CA). Ovation Pico WTA kit can successfully amplify samples with RIN numbers even as low as 2–5. For each array, 4 ug of cDNA was fragmented and biotin labeled using Encore Biotin module (NUGEN Technologies Inc, CA) and then added with hybridization control reagents (20× Eukaryotic controls and Control Oligo B2, Affymetrix Inc. CA). Samples were hybridized with Affymetrix Human Gene 2.0 ST arrays, incubated at 45°C for 18 hours rotating at 60 rpm in a GeneChip Hybridization oven (Affymetrix Inc, CA). Hybridized arrays were washed and stained with GeneChip HWS kit (Affymetrix Inc, CA) using Affymetrix 450 Fluidic Stations. Chips were scanned using Affymetrix GeneChip scanner 3000. To access the efficiency of cDNA synthesis, Poly A controls (dap, lys, phe, thr- Affymetrix Inc.) were spiked into the samples and hybridization controls (bioB, bioD, bioC and Cre, Affymetrix Inc.) were added to monitor labeling efficiency per the manufacturer’s instructions. Samples were processed as batches of 32 chips at a time. Raw files from Affymetrix Human Gene 2.0 array were preprocessed using RMA in R (Bioconductor oligo package, v.1.34.2), Supplementary Figure 1.¹² Raw and processed data were deposited in GEO ({"type":"entrez-geo","attrs":{"text":"GSE109278","term_id":"109278"}}GSE109278). Technical replicates were averaged for each sample and averages were used for further analysis by linear mixed models using the Bioconductor limma package (v.3.26.8).¹³ Pathway enrichment analyses were performed using the Fisher’s Exact Test on several databases (GO, Reactome, KEGG, WikiPathways). An adjusted p value of <0.05 was considered significant.