Quantification of regional DNA methylation by liquid chromatography/tandem mass spectrometry.
Anal Biochem
; 391(2): 106-13, 2009 Aug 15.
Article
in En
| MEDLINE
| ID: mdl-19442645
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Chromatography, High Pressure Liquid
/
DNA Methylation
/
Tandem Mass Spectrometry
Type of study:
Prognostic_studies
Limits:
Humans
Language:
En
Journal:
Anal Biochem
Year:
2009
Type:
Article
Affiliation country:
United States