Analysis of CpG island methylation using rolling Circle amplification (RCA) product microarray

Here we report a method for methylation analysis using rolling circle amplification (RCA) product microarray. We treated DNA samples with bisulfite and designed, for each CpG region in the target gene, a pair of padlock probes with one matching to the CpG sites derived from methylated CpG region, and the other matching to the TpG sites derived from their corresponding unmethylated allele. The padlock probes were hybridized to the PCR products of the bisulfite-treated genomic DNA, and were subsequently ligated to form single-strand, circular template for RCA reaction. The RCA products were immobilized on the slide to fabricate DNA microarray which hybridized a pair of universal dual-color probes to detect the methylation status of the CpG islands in the target gene. We tested the RCA product microarray with two tumor-related genes, P16 and IGFBP7, and successfully analyzed the methylation status of the CpG islands in the two genes. The microarray data were further confirmed by methylation-specific PCR analysis. Our results demonstrated that the RCA product microarray was hopeful for high-throughput detection of CpG island methylation.