Gene expression in tolerant and non-tolerant liver allografts

Liver biopsy samples were obtained as part of the LITMUS trial (ClinicalTrials.gov NCT02541916). All samples were obtained from liver transplant recipients prior to immunosuppression withdrawal. Percutaneous liver biopsies were performed under local anaesthesia using an 18-gauge Jamshidi needle. A 0.5cm portion of the biopsy to be used for gene expression was stored in RNAlater (Qiagen) for 24 hours at 4 °C and then transferred to -80 °C. Specimens were thawed and transferred to a clean, RNase free microcentrifuge tube. Tissue samples were incubated in 600 ul of lysis buffer (Ambion) with 6 ul of 2-mercaptoethanol for 10 minutes. Tissue samples were then homogenized using a nuclease-free disposable pellet pestle (Kimble Kontes) and processed using the PureLink RNA Mini Kit (Ambion). RNA quality was evaluated using an Bioanalyzer 2100 (Agilent) and samples with RIN > 7 were submitted for sequencing and analysis at The Center for Applied Genomics (Toronto, Canada). Overall design: Eight samples are from tolerant liver transplant recipients, and nine samples are from non-tolerant liver transplant recipients. All samples are liver biopsies obtained prior to immunosuppression withdrawal.