Aiba Toshiki, Saito Toshiyuki, Hayashi Akiko, Sato Shinji, Yunokawa Harunobu, Maruyama Toru, Fujibuchi Wataru, Kurita Hisaka, Tohyama Chiharu, Ohsako Seiichiroh
Laboratory of Environmental Health Sciences, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Department of Radiation Effects Research, National Institutes for Quantum and Radiological Science and Technology, 4-9-1 Anagawa, Inage-ku, Chiba, 263-8555, Japan.
BMC Mol Biol. 2017 Mar 9;18(1):7. doi: 10.1186/s12867-017-0083-2.
It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP.
Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans.
MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.
已经指出，环境因素或化学物质可导致起源于发育过程的疾病。为了检测DNA甲基化中异常的表观遗传改变，对于此类需要同时处理多个样本的研究，需要方便且经济高效的方法。我们在此介绍甲基化位点展示（MSD），这是一种制备DNA文库的独特技术。通过将其与扩增片段长度多态性（AFLP）分析相结合，我们开发了一种新方法，即MSD-AFLP。
甲基化位点展示文库仅由源自原始基因组DNA样本中5'端发生CpG甲基化的DNA片段的DNA组成。为了测试该方法的有效性，比较了小鼠肝脏、肾脏和海马组织中的CpG甲基化水平，以检查MSD-AFLP是否能检测到组织特异性差异甲基化CpG水平的细微差异。结果，检测到许多疑似组织特异性差异甲基化的CpG位点。鉴定了这些甲基化CpG位点附近的核苷酸序列，并通过甲基化敏感限制性内切酶（MSRE）-PCR分析确定甲基化水平，以确认AFLP分析的准确性。这些方法之间组织间甲基化水平的差异几乎相同。通过MSD-AFLP分析，我们检测到许多CpG位点，其组织特异性差异在统计学上小于5%，变异性小于10%。此外，MSD-AFLP分析可用于鉴定包括人类在内的其他生物体中的CpG甲基化位点。
MSD-AFLP分析有可能用于测量CpG甲基化水平的微小变化。鉴于MSD-AFLP分析研究具有显著的精度、灵敏度和通量，该方法在各种基于表观遗传学的研究中将具有优势。
