液相色谱-串联质谱法测定生物样本全基因组DNA甲基化

建立了基于液相色谱-电喷雾串联质谱的分析方法,对生物样本中全基因组DNA甲基化水平进行定量测定.首先将DNA从生物样本中提取出来,将DNA片段酶解为单核苷,利用液相色谱-串联质谱测定胞嘧啶核苷和5-甲基胞嘧啶核苷的含量,从而计算出其全基因组DNA甲基化率.利用该法研究了暴露于全氟辛烷磺酸的L-02细胞、10例原发性肝癌病例血浆样本和10例对照血浆样本的全基因组DNA甲基化水平,得出了它们的总甲基化率变化的初步结果.本方法操作简单,具有很高的灵敏度和稳定性,为研究生物样本,尤其是临床上易得但DNA含量极低的血浆样本的总甲基化水平提供了思路.

Determination of Global DNA Methylation in Biological Samples by Liquid Chromatography- Tandem Mass Spectrometry

A method based on liquid chromatography-electrospray ionization tandem mass spectrometry(LC-ESI-MS/MS) was developed to determine global DNA methylation level in biological samples.DNA was extracted from biological samples and digested by three enzymes into single nucleotides.Liquid chromatography coupled to tandem mass spectrometry was used to measure the concentrations of 2′-deoxycytidine and 2′-deoxy-5-methylcytidine respectively,so as to calculate the global DNA methylation ratios.The developed method was further used to explore the global DNA methylation level in normal human liver cell L-02 exposed to perfluorooctane sulfonate(PFOS) and plasma samples from 10 hepatocellular carcinoma patients and 10 control cases.This approach has high sensitivity and stability,and is easy to operate,enabling us to analyze the global DNA methylation level in various biological samples,especially those valuable samples(such as serum,plasma etc.) with extremely low concentration of DNA.