DNA methylation microarray screening
The DNAs of 15 donor livers carrying the CYP3A5*3/*3 genotype were extracted (approximately 500 ng of DNA per sample) and sent to Shanghai Jingzhou Genomics Technology Co., Ltd., where genome-wide DNA methylation was assessed using the Illumina Infinium Human Methylation850K BeadChip (Illumina Inc., USA) according to the manufacturer’s instructions. Array data (.IDAT files) were analysed using the ChAMP function in R for the determination of methylation levels. The methylation status of all probes was denoted as the β value, which is the ratio of the methylated probe intensity to the overall probe intensity (the sum of the methylated and unmethylated probe intensities plus the constant, α, where α = 100). CpG sites with |Δβ| ≥ 0.20 (in test vs. control) and adjustedP value ≤ 0.05, were considered to be differentially methylated sites. A CpG site was considered to be hypermethylated if its Δβ was ≥0.20, or hypomethylated if its Δβ was ≤ −0.20. The average β values for promoters and CpG islands were compared between Group1 (G1: low tacrolimus C0/D ratio group) and Group2 (G2: high tacrolimus C0/D ratio group). Promoters and CpG islands with |Δβ| ≥ 0.20 and adjusted P value ≤ 0.05, were retained for further analysis.
Pyrosequencing analysis
Genomic DNA (500 ng) extracted from the 23 CYP3A5*3/*3 genotype donor livers was transformed by sodium bisulfite using the EZ DNA Methylation Kit (Zymo Research, Orange County, California, USA) and further purified using the Wizard DNA Clean-up System (Promega, Madison, Wis, USA). The sequences of the primers used for the ABCB1 DNA methylation analysis are shown in Table 1 . Each PCR reaction system consisted of 100 ng of DNA converted by sodium bisulfite, 100 pM deoxyriboside triphosphate, 10 pM positive/reverse primers, and 1 unit of Taq polymerase (Merck KGaA, Darmstadt, Germany), which resulted in a final volume of 25 μL. After initial denaturation at 95 °C for 5 min, amplification was performed for 40 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, followed by a final elongation step at 72 °C for 5 min. PCR products were analysed by non-denatured 6% polyacrylamide gel electrophoresis, following staining with ethidium bromide.
In addition, genomic DNA was extracted from cells treated with the methylation inhibitor, 5-Aza-2-DC, and changes in ABCB1 DNA methylation were also evaluated by pyrosequencing.