Optimization of an HPLC Method for Determining the Genomic Methylation Levels of Taxus Cells.
An HPLC method for quantifying total DNA methylation in Taxus chinensis cells is described. Optimal conditions for the method were established as follows: DNA was hydrolyzed with DNA degradase at 37°C for 3 h. The mobile phase was a mixture of Solvent A [50 mM potassium dihydrogen phosphate/triethylamine (100:0.2, v/v)] and Solvent B (methanol); the gradient was 10% (v/v) solvent B. The calibration curves for deoxycytidine monophosphate (dCMP) and methylated dCMP were linear within 1.0-160.0 µg mL(-1), with correlation coefficients of 0.9996 and 0.9998. The limits of detection for dCMP and 5-mdCMP were 0.482 and 0.301 ng mL(-1), respectively, and the limits of quantification were 1.6 and 1.0 ng mL(-1), respectively. The method has been validated according to the current International Conference Harmonization guidelines. The method was able to quantify the content of dCMP and methylated dCMP specifically, accurately and precisely. The global DNA methylation level in different Taxus cells was measured using as little as 3 µg of DNA according to the optimized procedure. In addition, degradation of 5-methylcytosine was prevented.
Department of Biotechnology, College of Life Science and Technology, Institute of Resource Biology and Biotechnology, Huazhong University of Science and Technology, Wuhan 430074, China.
Xiao-li
XL
Li
Department of Biotechnology, College of Life Science and Technology, Institute of Resource Biology and Biotechnology, Huazhong University of Science and Technology, Wuhan 430074, China.
Jie
J
Yuan
Department of Biotechnology, College of Life Science and Technology, Institute of Resource Biology and Biotechnology, Huazhong University of Science and Technology, Wuhan 430074, China.
Yan-shan
YS
Dong
Department of Biotechnology, College of Life Science and Technology, Institute of Resource Biology and Biotechnology, Huazhong University of Science and Technology, Wuhan 430074, China Key Laboratory of Molecular Biophysics of Ministry of Education, Wuhan 430074, China fuch2003@126.com.
Chun-hua
CH
Fu
Department of Biotechnology, College of Life Science and Technology, Institute of Resource Biology and Biotechnology, Huazhong University of Science and Technology, Wuhan 430074, China Key Laboratory of Molecular Biophysics of Ministry of Education, Wuhan 430074, China Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains, Huanggang, China.
Mao-Teng
MT
Li
Department of Biotechnology, College of Life Science and Technology, Institute of Resource Biology and Biotechnology, Huazhong University of Science and Technology, Wuhan 430074, China Key Laboratory of Molecular Biophysics of Ministry of Education, Wuhan 430074, China.
Long-jiang
LJ
Yu
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019175:DNA Methylation; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D023281:Genomics; D020946:Taxus / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D018744:DNA, Plant