Establishment and application of medical method for quantification of genomic DNA methylation in methotrexate drug resistance cells
10.3760/cma.j.issn.1009-9158.2009.02.026
- VernacularTitle:氨甲蝶呤耐药细胞内整体DNA甲基化水平检测方法的建立及其应用
- Author:
Ming LI
;
Shilian HU
;
Xiaodong HE
;
Shaoneng TAO
;
Lin DONG
;
Yuanyuan ZHU
;
Jianfeng WU
;
Zuojun SHEN
- Publication Type:Journal Article
- Keywords:
Methotrexate;
DNA methylation;
Electrophoresis,capillary
- From:
Chinese Journal of Laboratory Medicine
2009;32(2):204-208
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a rapid and convenient method for determination of genomic DNA methylation in cells.Methods Five standard substances (dC, mdC, dA, dT and dG) were separated by high-performance capillary electrophoresis.Bare fused-silica capillary was used and eletrophoresis buffer was 48 mmol/L NaHCO3 with 60 mmol/L SDS, pH 9.6.The temperature of separation was controlled at 25 ℃ and a voltage of 20 kV was applied.The separation of the mixture was performed at a wavelength of 256 nm with UV-Vis detection and injection time was 5 seconds at 0.7 psi.Under optimal condition,genomic DNA methylation in methotrexate drug-resistant A549 cells was detected.Results The optimal condition was made by adjusting SDS concentration(40, 60, 80 mmol/L), pH value of running buffer(9.4,9.6, 9.8), voltage(15, 17, 19, 20, 22 kV), injection time(5, 10, 15, 20, 30 s) and capillary temperature(15, 20, 25, 30 ℃).The method for determination of genomic DNA methylation in cells was established.Five substances were completely separated by high-performance capillary electrophoresis in 10 mins.Intra-day coefficient of variation was less than 0.2% and inter-day coefficient of variation was less than 2%.The minimal detection limit was 2 μmol/L.Percentage of mdC in A549 parent cells was (4.80 ±0.52) %.Percentage of mdC in 15, 30, 40 μmol/L methotrexate drug-resistant A549 cells were (4.20±0.44) %, ( 3.70 ± 0.36 ) %, (3.10±0.35 ) %, respectively.Conclusions Genomic DNA methylation can be quantificated by high-performance capillary electrophoresis efficiently, rapidly, conveniently and sensitively.Genomic DNA methylation in methotrexate drug resistance cells decreases significantly.
