title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.
abstract: 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.
authors.affiliation: 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.
authors.forename: Toshiki
authors.initials: T
authors.lastname: Aiba
authors.affiliation: Department of Radiation Effects Research, National Institutes for Quantum and Radiological Science and Technology, 4-9-1 Anagawa, Inage-ku, Chiba, 263-8555, Japan. saito.toshiyuki@qst.go.jp.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Saito
authors.affiliation: Department of Radiation Effects Research, National Institutes for Quantum and Radiological Science and Technology, 4-9-1 Anagawa, Inage-ku, Chiba, 263-8555, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Hayashi
authors.affiliation: Maze, Inc., 1-2-17 Sennincho, Hachioji-shi, Tokyo, 193-0835, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Sato
authors.affiliation: Maze, Inc., 1-2-17 Sennincho, Hachioji-shi, Tokyo, 193-0835, Japan.
authors.forename: Harunobu
authors.initials: H
authors.lastname: Yunokawa
authors.affiliation: Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.|Department of Life Science & Medical Bioscience, Graduate School of Advanced Science & Engineering, Waseda University, Tokyo, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Maruyama
authors.affiliation: Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Fujibuchi
authors.affiliation: 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.|Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, Gifu, Japan.
authors.forename: Hisaka
authors.initials: H
authors.lastname: Kurita
authors.affiliation: 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.|Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: Chiharu
authors.initials: C
authors.lastname: Tohyama
authors.affiliation: 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. ohsako@m.u-tokyo.ac.jp.
authors.forename: Seiichiroh
authors.identifier: 0000-0001-8237-7992
authors.initials: S
authors.lastname: Ohsako
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis / Q000191:economics / Q000379:methods*; D000818:Animals; D018899:CpG Islands; D004247:DNA / Q000235:genetics; D019175:DNA Methylation; D008297:Male; D008810:Mice, Inbred C57BL
chemical_list: D004247:DNA
keywords: AFLP; DNA methylation profiling; Epigenetics