sample preparation: Participant baseline evaluation included an ultrasound-guided percutaneous liver biopsy yielding two cores, which was completed on all participants except for those with a contraindication (e.g., anticoagulation). The first core was fixed in formalin, and subsequently underwent histopathologic review by a single expert pathologist blinded to treatment (D.E.K., National Institutes of Health). Histological scoring, including NAFLD Activity Score (NAS) and fibrosis stage, was performed according to the Nonalcoholic Steatohepatitis Clinical Research Network scoring system.(4) The second core was placed in an RNA stabilization reagent (RNAlater, Qiagen) and stored at -80o C for gene expression analysis. Both 1H-MRS and liver biopsy were repeated at 12 months following randomization. A total of 19 participants randomized to tesamorelin and 24 participants randomized to placebo had paired liver specimens available for gene sequencing. Liver cores were placed in an RNA stabilization reagent (RNAlater, Qiagen) and stored at -80 C for gene expression analysis Following extraction from liver tissue using RNeasy Plus Mini Kit (Qiagen), total RNA was quantified using the Quant-iT™ RiboGreen® RNA Assay Kit and normalized to 5ng/ul. Following plating, 2 uL of ERCC controls (using a 1:1000 dilution) and a k562 control were spiked into each sample. An 200ng aliquot of each sample was taken for library preparation using the Illumina TruSeq™ Stranded mRNA Sample Preparation Kit. oligo dT beads were used to select mRNA from the total RNA sample, followed by heat fragmentation and cDNA synthesis from the RNA template. The resultant 400bp cDNA underwent dual-indexed library preparation: 'A' base addition, adapter ligation using P7 adapters, and PCR enrichment using P5 adapters. After enrichment the libraries were quantified using Quant-iT PicoGreen (1:200 dilution).
GEO Accession
GSM4520842