A Novel Microarray-based Method For Quantifying DNA Methylation Levels Of CD11a (ITGAL) And CD70 (TNFSF7)

Objective:To establish an methylation-specific oligonucleotide microarray for detecting the methylation status of CpG sites which change significantly in CD11a and CD70 genes of SLE T cells.Methods:1. Generate a standard curve from fully methylated and fully unmethylated DNA samples:two kinds of labeled PCR products were mixed in five proportions, so we get a gradient proportion of methylated products at 100%,75%,50%,25% and 0% as reference standard as M,and U for unmethylated ones,use these data,we can draw a standard curve with intensity ratio of M/(M+U) as the ordinate and the percentage of methylated products in the mixture as the abscissa,we can get five scatter spots for each pair of probes.establish a one-dimensional linear regression.2. Measure methylation level in CD70 and CDlla promoter fragments in CD4+ T cell DNA samples from one patient with SLE and one healthy control. The mean methylation level was compared to that measured by bisulfite sequencing of 20 clones from the same samples.3. Isolation of CD4+ T cells from peripheral blood of 20 cases SLE patients and 20 healthy controls (in the premise of informed consent), and then extracting DNA and bisulfite conversion. The interested segments was amplified by nest PCR.The PCR products were hybridized with the microarray, and then the image was scaned. The methylation degree was calculated.Results:1. Microarray results of fully methylated and fully unmethylated DNA samples were in accordence with bisulfite genomic sequencing, the standard curve of five CpG sites take on linear relationship, R2= 0.98 (CD11a); R2= 0.99 (CD70).2. The microarray prediction was found to be highly accurate when compared to bisulfite sequencing.3. The results confirmed that methylation levels of CD11a and CD70 promoter regions in SLE CD4+ T cells were significantly lower compared to healthy controls, and the microarrays were able to detect differences in the methylation status between SLE patient and healthy control samples.Conclusion:These results indicate that our new microarray-based assay could prove to be a highly reliable, rapid and cost-effective test for disease status and prognostic of SLE.