Australian Chronic Allograft Dysfunction III

The Australian Chronic Allograft Dysfunction (AUSCAD) study is an ongoing single centre cohort study at Westmead hospital in Australia. In this section of the study, we aimed to identify biomarkers for allograft rejection in kidney transplant recipients, 3-months after their transplant. Our study recruited 123 patients, each having protocol renal allograft biopsies taken 3-months post transplantation. Overall design: 123 patients had renal allograft biopsies collected 3 months post-transplant at Westmead hospital. One core was used for sequencing, the other was analysed by a single pathologist at Westmead. The banff19 classification system was used for diagnosis. For the purpose of this study, a combined criteria of ABMR and TCMR was classified at Rejection (Rej). Biopsies with no evidence of rejection were treated as the control group (Nill). Kidney biopsy specimens were left in RNAlater (Sigma-Aldrich) overnight at 4°C before removal and storage at -80°C until RNA extraction. Specimens were chemically and physically lysed by using 2-Mercaptoethanol (Sigma-Aldrich) and TissueLyser II (Qiagen, Germany), follow by RNA extraction using AllPrep DNA/RNA/microRNA and MiniElute clean- up kits (Qiagen, Germany). All RNA samples were frozen and stored at -80°C and then sent in bulk to the Australian Genome Research Facility (AGRF, Melbourne, Australia). Sample QC and library preparation were performed in-house, and the resultant libraries sequenced using the NovaSeq 6000 platform (Illumina)