Abstract
DNA methylation in eukaryotes is the key event that governs epigenetic phenomena, including development and cell differentiation. Its collapse results in disorders such as cancer, aging and so on. Therefore it is essential to find a comprehensive method for detecting the change of methylation sites, for both basic and clinical research. Compared with gene expression profiling analysis, the analysis of methylation has several advantages. 1) It is rather easy to detect low-abundance transcripts. In gene expression profiling, detecting rarely-expressed genes is usually quite hard. 2) It is possible to identify unknown transcripts, including novel types of transcripts such as poly(A)-less ones. 3) It is possible to understand the epigenetic mechanisms. Recently we proposed a new highly sensitive, high-coverage method using a methylation-sensitive restriction enzyme in combination with the selective PCR of HiCEP. This enables us to analyze approximately 30,000 methylation sites simultaneously. In the present study, we examine whether this procedure accurately detects changes in methylation using bisulfate sequencing analysis, and whether the methylation closely correlates with the expression of adjacent genes. This is an ideal fragment analysis system since it is simple, has few peaks and few false signals. Peak simulation using sequence information in the public database seems to be realistic. We used this approach and evaluate its reliability. We also discuss a protocol for the analysis using several hundred cells.
