Zinellu Angelo, Sotgiu Elisabetta, Assaretti Stefano, Sotgia Salvatore, Paliogiannis Panagiotis, Pintus Gianfranco, Mangoni Arduino A, Carru Ciriaco
Department of Biomedical Sciences, University of Sassari, Sassari, Italy.
Department of Clinical and Experimental Medicine, University of Sassari, Sassari, Italy.
J Anal Methods Chem. 2017;2017:4065892. doi: 10.1155/2017/4065892. Epub 2017 Dec 3.
Alterations in global DNA methylation are implicated in various pathophysiological processes. The development of simple and quick, yet robust, methods to assess DNA methylation is required to facilitate its measurement and interpretation in clinical practice. We describe a highly sensitive and reproducible capillary electrophoresis method with UV detection for the separation and detection of cytosine and methylcytosine, after formic acid hydrolysis of DNA extracted from human whole blood. Hydrolysed samples were dried and resuspended with water and directly injected into the capillary without sample derivatization procedures. The use of a run buffer containing 50 mmol/L BIS-TRIS propane (BTP) phosphate buffer at pH 3.25 and 60 mmol/L sodium acetate buffer at pH 3.60 (4 : 1, ) allowed full analyte identification within 11 min. Precision tests indicated an elevated reproducibility with an interassay CV of 1.98% when starting from 2 μg of the extracted DNA. The method was successfully tested by measuring the DNA methylation degree both in healthy volunteers and in reference calf thymus DNA.
全基因组DNA甲基化的改变与多种病理生理过程有关。需要开发简单、快速且可靠的方法来评估DNA甲基化，以便在临床实践中进行测量和解读。我们描述了一种高灵敏度且可重复的毛细管电泳方法，该方法采用紫外检测，用于在对从人全血中提取的DNA进行甲酸水解后，分离并检测胞嘧啶和甲基胞嘧啶。水解后的样品经干燥后用水重悬，无需样品衍生化程序即可直接注入毛细管。使用含有50 mmol/L BIS-TRIS丙烷（BTP）磷酸盐缓冲液（pH 3.25）和60 mmol/L乙酸钠缓冲液（pH 3.60，比例为4:1）的运行缓冲液，可在11分钟内实现对所有分析物的完全鉴定。精密度测试表明，从2 μg提取的DNA开始时，批间变异系数为1.98%，重现性较高。该方法通过测量健康志愿者和参考小牛胸腺DNA中的DNA甲基化程度得到了成功验证。
