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著作: Husseiny Mohamed I/[黒田 暁生]/Kaye Alexander N/Nair Indu/Kandeel Fouad/Ferreri Kevin/Development of a quantitative methylation-specific polymerase chain reaction method for monitoring beta cell death in type 1 diabetes./[PLoS ONE]

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EID
260654
EOID
777126
Map
0
LastModified
2015年5月11日(月) 15:35:18
Operator
松井 栄里
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TRUE
Censor
0
Owner
黒田 暁生
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種別 必須 学術論文(審査論文)
言語 必須 英語
招待 推奨
審査 推奨
カテゴリ 推奨
共著種別 推奨
学究種別 推奨
組織 推奨
著者 必須
  1. (英) Husseiny Mohamed I
    役割 任意
    貢献度 任意
    学籍番号 推奨
  2. 黒田 暁生([徳島大学.先端酵素学研究所.基幹研究部門])
    役割 任意
    貢献度 任意
    学籍番号 推奨
  3. (英) Kaye Alexander N
    役割 任意
    貢献度 任意
    学籍番号 推奨
  4. (英) Nair Indu
    役割 任意
    貢献度 任意
    学籍番号 推奨
  5. (英) Kandeel Fouad
    役割 任意
    貢献度 任意
    学籍番号 推奨
  6. (英) Ferreri Kevin
    役割 任意
    貢献度 任意
    学籍番号 推奨
題名 必須

(英) Development of a quantitative methylation-specific polymerase chain reaction method for monitoring beta cell death in type 1 diabetes.

副題 任意
要約 任意

(英) DNA methylation is a mechanism by which cells control gene expression, and cell-specific genes often exhibit unique patterns of DNA methylation. We previously reported that the mouse insulin-2 gene (Ins2) promoter has three potential methylation (CpG) sites, all of which are unmethylated in insulin-producing cells but methylated in other tissues. In this study we examined Ins2 exon 2 and found a similar tissue-specific methylation pattern. These methylation patterns can differentiate between DNA from insulin-producing beta cells and other tissues. We hypothesized that damaged beta cells release their DNA into circulation at the onset of type 1 diabetes mellitus (T1DM) and sought to develop a quantitative methylation-specific polymerase chain reaction (qMSP) assay for circulating beta cell DNA to monitor the loss of beta cells. Methylation-specific primers were designed to interrogate two or more CpG in the same assay. The cloned mouse Ins2 gene was methylated in vitro and used for development of the qMSP assay. We found the qMSP method to be sensitive and specific to differentiate between insulin-producing cells and other tissues with a detection limit of 10 copies in the presence of non-specific genomic DNA background. We also compared different methods for data analysis and found that the Relative Expression Ratio method is the most robust method since it incorporates both a reference value to normalize day-to-day variability as well as PCR reaction efficiencies to normalize between the methylation-specific and bisulfite-specific components of the calculations. The assay was applied in the streptozotocin-treated diabetic mouse model and detected a significant increase in circulating beta cell DNA before the rise in blood glucose level. These results demonstrate that this qMSP assay can be used for monitoring circulating DNA from insulin-producing cells, which will provide the basis for development of assays to detect beta cell destruction in early T1DM.

キーワード 推奨
  1. (英) Animals
  2. (英) Base Sequence
  3. (英) Cell Death
  4. (英) DNA
  5. (英) DNA Methylation
  6. (英) Diabetes Mellitus, Type 1
  7. (英) Exons
  8. (英) Insulin
  9. (英) Insulin-Secreting Cells
  10. (英) Mice
  11. (英) Mice, Inbred BALB C
  12. (英) Mice, Inbred NOD
  13. (英) Mice, SCID
  14. (英) Molecular Sequence Data
  15. (英) Polymerase Chain Reaction
  16. (英) Reproducibility of Results
  17. (英) Sequence Homology, Nucleic Acid
発行所 推奨
誌名 必須 PLoS ONE(Public Library of Science)
(eISSN: 1932-6203)
ISSN 任意 1932-6203
ISSN: 1932-6203 (eISSN: 1932-6203)
Title: PloS one
Title(ISO): PLoS One
Publisher: Public Library of Science
 (NLM Catalog  (Scopus  (CrossRef (Scopus information is found. [need login])
必須 7
必須 10
必須 e47942 e47942
都市 任意
年月日 必須 2012年 10月 29日
URL 任意
DOI 任意 10.1371/journal.pone.0047942    (→Scopusで検索)
PMID 任意 23144715    (→Scopusで検索)
NAID 任意
WOS 任意 000310705300026
Scopus 任意 2-s2.0-84868113134
評価値 任意
被引用数 任意
指導教員 推奨
備考 任意
  1. (英) Affiliation: Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of City of Hope, Duarte, California, United States of America.

  2. (英) PublicationType: Journal Article

  3. (英) PublicationType: Research Support, Non-U.S. Gov't