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High-throughput and cost-effective global DNA methylation assay by liquid chromatography-mass spectrometry

Reputable Mentor II
Reputable Mentor II
Li X, Franke AA.
Anal Chim Acta. 2011 Oct 3;703(1):58-63.
An affordable and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the accurate and precise determination of global DNA methylation levels in peripheral blood. Global DNA methylation extent was expressed as the ratio of methylated 2'-deoxycytidine (5MedC) to 2'-deoxyguanosine (dG), which were obtained after DNA extraction and hydrolysis and determined by positive electrospray LC-ESI-MS/MS. The cost-effective internal standards (15)N(3)-dC and (15)N(5)-dG were incorporated for the accurate quantification of 5MedC and dG, respectively. The desired nucleoside analytes were separated and eluted by LC within 2.5 min on a reverse phase column with a limit of detection of 1.4femtomole on column for 5MedC. Sample preparation in 96-well format has significantly increased the assay throughput and filtration was found to be a necessary step to assure precision. Precision was performed with repeated analysis of four DNA QC sample over 12 days, with mean intra- and inter-day CVs of 6% and 11%, respectively. Accuracy was evaluated by comparison with a previously reported method showing a mean CV of 4% for 5 subjects analyzed. Furthermore, application of the assay using a benchtop orbitrap LCMS in exact mass full scan mode showed comparable sensitivity to tandem LCMS using multiple reaction monitoring.
University of Hawaii Cancer Center, Honolulu, 96813, USA.
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‎10-15-2021 05:28 AM
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