Ncounter digital analyzer system

All NASH subjects with sufficient remaining formalin-fixed, paraffin-embedded (FFPE) hepatic tissue for RNA extraction (n=47) were included for gene expression profiling. Briefly, total RNA was extracted from five 5-μm-thick FFPE tissue sections using the High Pure RNA paraffin kit (Roche). Gene expression profiling was performed using 100–400ng total RNA by using nCounter Digital Analyzer system (NanoString) according to the manufacturer’s instructions. Raw transcript count data were log transformed and scaled by geometric mean of control probe data by using NanoString normalizer module implemented in GenePattern genomic analysis toolkit (www.broadinstitute.org/cancer/software/genepattern). Gene expression dataset is available at National Center for Biotechnology Information Gene Expression Omnibus database (accession number: GSE 69248).

The 186-gene signature was implemented in the digital transcript counting (nCounter) assay (NanoString). Expression profiling was performed with 100ng to 400ng total RNA by using nCounter Digital Analyzer system (NanoString) according to manufacturer's instruction. For the analysis of the training cohort, the first generation of reagent plate (“white” Prep Plate) was used. For the validation cohort, newer version (“green” New Prep Plate) with improved sensitivity for signal detection was used. Poor quality profiles were detected based on maximum signal intensity from positive control probes <8,000U for the older reagent plate, and median signal intensity >100U for the newer reagent plate according to manufacturer’s recommendation. Raw transcript count data were log-transformed and scaled by geometric mean of control probe data by using NanoString normalizer module implemented in GenePattern genomic analysis toolkit (www.broadinstitute.org/genepattern). Genome-wide expression profiling for paired biopsies and explanted liver was performed by using whole-genome DASL assay (Illumina) according to manufacturer's instruction. Scanned data were extracted by Genome Studio software ver.3 (Illumina), and normalized by cubic spline algorithm implemented in GenePattern Illumina Normalizer module as previously described.^{18 (link)}