亲水作用色谱法测定组织中全基因组DNA甲基化水平

Determination of global DNA methylation in tissues by hydrophilic interaction liquid chromatography

建立了亲水作用色谱（HILIC）测定组织中全基因组DNA甲基化水平的方法。采用苯酚-氯仿提取组织中的DNA,提取的DNA用88%甲酸在140℃下裂解,经N2吹干后,加乙腈-水（9∶1,v/v）溶解,用Waters BEH HILIC柱进行分离,在277nm波长下检测胞嘧啶（Cyt）及5-甲基胞嘧啶（5-mCyt）含量。结果表明,以乙腈-10mmol/L甲酸铵溶液（94∶6,v/v）为流动相,流速为0.5mL/min,Cyt与5-mCyt分离较好,保留时间分别为2.6与3.1min。胞嘧啶的线性范围为1~900μmol/L,相关系数为0.999 9;5-甲基胞嘧啶的线性范围为1~64μmol/L,相关系数为0.999 8。胞嘧啶和5-甲基胞嘧啶的检出限为54nmol/L（柱中为0.54pmol）,定量限为250nmol/L（柱中为2.5pmol）;在5~900μmol/L的添加水平下,胞嘧啶和5-甲基胞嘧啶的平均加标回收率为94.7%~100.5%,相对标准偏差小于1.48%。用该方法检测了结肠癌组织中DNA甲基化水平,结果显示该癌组织中全基因组的DNA甲基化均值为4.0%。该方法快速、简单,稳定性好,灵敏度较高,能满足全基因组DNA甲基化的检测要求。

A hydrophilic interaction liquid chromatographic（HILIC） method has been developed for the determination of global DNA methylation in tissues.The DNA was extracted by phenol-chloroform,hydrolyzed with 88% formic acid at 140 ℃,evaporated under nitrogen at 60 ℃,and reconstituted in a mixture of acetonitrile/water（90∶10,v/v）;the separation was achieved on a Waters bridged ethylene hybrid（BEH） HILIC column（100 mm×2.1 mm,1.7 μm）.The cytosine（Cyt） and 5-methylcytosine（5-mCyt） were separated in a fairly short time（3.5 min） by isocratic elution with a mixture of acetonitrile/10 mmol/L ammonium formate（94∶6,v/v） as the mobile phase.Under the optimized conditions,calibration standard curve showed a good linearity in the range 1-900 μmol/L for Cyt and in the range 1-64 μmol/L for 5-mCyt with correlation coefficients of 0.9999 and 0.9998,respectively.The limit of detection（LOD） was 54 nmol/L（0.54 pmol on-column） both for Cyt and 5-mCyt,and the limit of quantification was 250 nmol/L（2.5 pmol on-column） both for Cyt and 5-mCyt.The recoveries of Cyt at the spiked levels of 90,450,900 μmol/L and 5-mCyt at the spiked levels of 5,16,64 μmol/L all ranged from 94.7% to 100.5% with a relative standard deviations less than 1.48%.The method was applied to the analysis of DNA from colon cancer tissue,and the average degree of methylation was 4.0%.The method is rapid,simple,sensitive,reliable,and suitable for the determination of global DNA methylation.