Comparative Landscape of Small RNAs in Tissue and Liquid Biopsies for Liver Transplant Outcomes [Perfusate small RNAseq]

Ischemia-reperfusion injury (IRI) is an inevitable consequence of liver transplantation, arising during donor organ procurement and reoxygenation. Severe IRI is a leading contributor to early allograft dysfunction (EAD), a post-transplant complication associated with reduced graft survival. Current postoperative biomarkers provide limited time for intervention, highlighting a need to identify preoperative biomarkers of IRI. Meanwhile, tRNA fragments (tRFs) have emerged as novel biomarkers in various diseases, but remain unexplored in the context of liver transplant. We performed small RNA sequencing on donor liver biopsies to investigate IRI-associated transcript changes. In parallel, donor liver perfusates were analyzed as a non-invasive surrogate for tissue profiling. Overall design: Liver tissue and perfusate samples were obtained from adult orthotopic liver transplant (OLT) recipients (?18 years old) at Methodist University Hospital after obtaining informed consent. All procedures were conducted under protocols approved by the University of Tennessee Health Science Center Institutional Review Board (IRB#20-07742-XM and IRB#20-07640-XP). Participants in the LUT cohort were recruited between September 2018 and March 2020, and those in the UTL cohort were recruited between December 2020 and December 2022. Needle biopsy specimens (L1: pre-implantation; L2: post-reperfusion) were immediately immersed in RNAlater (Thermo Fisher Scientific, Cat. #AM7021) and incubated at 4 °C overnight to ensure complete permeation. RNAlater was then removed, and the samples were stored at ?80 °C until RNA extraction. Perfusate samples were collected at the end of organ procurement concurrently with pre-implantation liver (L1) biopsies. The solutions were centrifuged at 2,400 rpm for 30 minutes at room temperature to remove debris. The resulting supernatant was aliquoted and stored at ?80 °C until RNA isolation. Patients were grouped based on transplant outcome, measured by day 1 AST (aspartate aminotransferase) values. EAD (early allograft dysfunction): day 1 AST more than 2000; NF (normal function): day 1 AST less or equal to 2000.