Analysis of methylation level of bone marrow mesenchymal stemcells during senescence by ultra performance liquidchromatography-tandem mass spectrometry
10.3969/j.issn.1001-1978.2017.09.021
- VernacularTitle:超高效液相色谱-串联质谱法分析骨髓间充质干细胞衰老过程中的全基因组DNA甲基化水平变化
- Author:
Guodong HE
;
Xiangyu YANG
;
Xiaohong LI
;
Yu PAN
;
Xiyong YU
- Keywords:
ultra performance liquid chromatography;
mass spectrometry;
bone marrow mesenchymal stem cells;
senescence;
DNA methylation;
enzymatic hydrolysis
- From:
Chinese Pharmacological Bulletin
2017;33(9):1298-1303
- CountryChina
- Language:Chinese
-
Abstract:
Aim To measure the methylation rate of bone marrow mesenchymal stem cells (BMMSCs) using ultra-performance liquid chromatography-tandem mass spectrometry, and determine the methylation rate in the process of senescence.Methods The DNA extracted from BMMSCs would be digested into individual deoxynucleosides using enzymatic hydrolysis.To quantify the global genomic DNA methylation rate,we developed a method using ultra-performance liquid chromatography-tandem mass spectrometry with multiple reaction monitoring to simultaneously measure the levels of 5-methyl deoxycytidine and deoxyguanosine in digested genomic DNA.Results The DNA methylation rate could be analyzed within two minutes after hydrolyzing 1μg DNA for an hour.The detection limit of DNA methylation rate was 5.00×10-4.The coefficient of variance of the intra-day and inter-day precision was within 7.12×10-2 and 0.119, respectively.With the subculture of BMMSCs, the methylation rate gradually decreased.The methylation rate of stem cells was the lowest in 4~6 generation, and gradually increased with the subculture.Conclusions Using this method, the preliminary data on DNA methylation of BMMSCs in the process of senescence are obtained.The method is simple and convenient for sample preprocessing, and the method is fast and accurate with good sensitivity and repeatability.
