摘要
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 0.9999 and 0.9998,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.
出处
《色谱》
CAS
CSCD
北大核心
2011年第4期342-345,共4页
Chinese Journal of Chromatography
基金
广东医学院博士基金
关键词
亲水作用色谱
全基因组DNA甲基化
组织
结肠癌
hydrophilic interaction liquid chromatography(HILIC)
global DNA methylation
tissues
colon cancer