Open AccessOptimization of an HPLC Method for Determining the Genomic Methylation Levels ofTaxusCells
Author(s) -
Xiaoli Li,
Jing Yuan,
Yanshan Dong,
Chunhua Fu,
Maoteng Li,
Liang Yu
Publication year - 2015
Publication title -
journal of chromatographic science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.362
H-Index - 56
eISSN - 1945-239X
pISSN - 0021-9665
DOI - 10.1093/chromsci/bmv129
Subject(s) - chemistry , chromatography , methylation , taxus , high performance liquid chromatography , biochemistry , botany , dna , biology
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.