色谱

色谱 ›› 2011, Vol. 29 ›› Issue (04): 342-345.DOI: 10.3724/SP.J.1123.2011.00342

• 特别策划 • 上一篇    下一篇

亲水作用色谱法测定组织中全基因组DNA甲基化水平

张良滔, 张立坚, 张俊杰, 刘春安, 蔡春*   

  1. 广东医学院, 广东 湛江 524023
  • 收稿日期:2010-12-22 修回日期:2011-01-23 出版日期:2011-04-28 发布日期:2011-04-28
  • 通讯作者: 蔡春,教授,研究方向为生物医学分析. E-mail: caichun@gdmc.edu.cn
  • 基金资助:

    广东医学院博士基金.

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

ZHANG Liangtao, ZHANG Lijian, ZHANG Junjie, LIU Chunan, CAI Chun*   

  1. Guangdong Medical College, Zhanjiang 524023, China
  • Received:2010-12-22 Revised:2011-01-23 Online:2011-04-28 Published:2011-04-28

PDF

511

被引次数

5

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277 nm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-10 mmol/L甲酸铵溶液(94:6, v/v)为流动相,流速为0.5 mL/min, Cyt与5-mCyt分离较好,保留时间分别为2.6与3.1 min。胞嘧啶的线性范围为1～900 μmol/L,相关系数为0.9999; 5-甲基胞嘧啶的线性范围为1～64 μmol/L,相关系数为0.9998。胞嘧啶和5-甲基胞嘧啶的检出限为54 nmol/L(柱中为0.54 pmol),定量限为250 nmol/L(柱中为2.5 pmol);在5～900 μmol/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。

关键词: 结肠癌, 亲水作用色谱, 全基因组DNA甲基化, 组织

Abstract: 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.

Key words: colon cancer, global DNA methylation, tissues, hydrophilic interaction liquid chromatography (HILIC)