Quantification of regional DNA methylation by liquid chromatography/tandem mass spectrometry

Zhongfa Liu, Jiejun Wu, Zhiliang Xie, Shujun Liu, Patty Fan-Havard, Tim H.M. Huang, Christoph Plass, Guido Marcucci, Kenneth K. Chan

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Promoter hypermethylation-associated tumor suppressor gene (TSG) silencing has been explored as a therapeutic target for hypomethylating agents. Promoter methylation change may serve as a pharmacodynamic endpoint for evaluation of the efficacy of these agents and predict the patient's clinical response. Here a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been developed for quantitative regional DNA methylation analysis using the molar ratio of 5-methyl-2′-deoxycytidine (5mdC) to 2′-deoxycytidine (2dC) in the enzymatic hydrolysate of fully methylated bisulfite-converted polymerase chain reaction (PCR) amplicons as the methylation indicator. The assay can differentiate 5% of promoter methylation level with an intraday precision ranging from 3 to 16% using two TSGs: HIN-1 and RASSF1A. This method was applied to characterize decitabine-induced promoter DNA methylation changes of these two TSGs in a breast cancer MCF-7 cell line. Promoter methylation of these TSGs was found to decrease in a dose-dependent manner. Correspondingly, the expression of these TSGs was enhanced. The sensitivity and reproducibility of the method make it a valuable tool for specific gene methylation analysis that could aid characterization of hypomethylating activity on specific genes by hypomethylating agents in a clinical setting.

Original languageEnglish (US)
Pages (from-to)106-113
Number of pages8
JournalAnalytical Biochemistry
Volume391
Issue number2
DOIs
StatePublished - Aug 15 2009
Externally publishedYes

Keywords

  • LC-MS/MS
  • Quantification
  • Regional DNA methylation

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  • Cite this

    Liu, Z., Wu, J., Xie, Z., Liu, S., Fan-Havard, P., Huang, T. H. M., Plass, C., Marcucci, G., & Chan, K. K. (2009). Quantification of regional DNA methylation by liquid chromatography/tandem mass spectrometry. Analytical Biochemistry, 391(2), 106-113. https://doi.org/10.1016/j.ab.2009.05.012