超高效液相色谱-荧光法检测精液中DNA甲基化水平

Ultra-high performance liquid chromatography-fluorescence method for determination of DNA methylation levels in human semen

目的建立超高效液相色谱-荧光法（UPLC-FLD）定量分析精液标本中DNA甲基化水平。方法精液样本DNA提取、水解后,以100 mmol/L 2-溴苯乙酮为衍生试剂,在0.51 mol/L乙酸乙腈溶液中80℃加热60 min,生成脱氧胞嘧啶核苷（d C）和5-甲基脱氧胞嘧啶核苷（5md C）的荧光衍生物（λex=306 nm,λem=378 nm）。取1μL上清液进样,经Agilent C18色谱柱（2.1×100 mm、1.8μm）分离[流动相为28%甲醇＋10%乙腈＋62%水溶液（含0.1%甲酸）,柱温40℃,流速0.3 m L/min]及统计学分析,并用临床样本进行验证。结果在上述最佳衍生反应与色谱分离条件下,7 min即可将精液DNA中的d C与5md C完全分离,无需消除RNA干扰;本法中5md C的检测限为2.5 pg/μL,线性范围为0.025-0.75 ng/μL,r〉0.99,日间、日内变异系数（CV）〈5.6%,衍生化合物室温条件放置30 h内比较稳定。临床精液样本结果表明,少弱畸精子症患者的DNA甲基化水平低于健康男性。结论建立的UPLC-FLD法可快速、简便、准确、稳定地检测基因组DNA甲基化水平。

Objective To establish a ultra-high performance liquid chromatography-fluorescence detection（ UPLC-FLD） for quantification of global DNA methylation in human semen. Methods The derivatization reaction of hydrolyzed semen DNA with 2-bromoacetophenone（ 100 mmol / L） was heated at 80 ℃ for 60 min in 0. 51 mol / L of acetic acid acetonitrile solution to generate the fluorescent products of deoxycytidine（ d C） and 5-methyl cytosine nucleoside（ 5md C）（ λex= 306 nm,λem= 378 nm）. One microliter of supernatant was passed through a C18 chromatographic column with the following experimental conditions： mobile phase： 28% methanol,10%acetonitrile and 62% water（ plus 0. 1% formic acid）,flow rate： 0. 3 m L / min,column temperature： 40 ℃,injection volume： 1 μL.The derivatization products was eluted and statistical analysis was performed. The methylation of DNA in clinical semen samples was then determined and verified by the established method. Results Under the given optimum conditions,d C and 5md C could be isolated completely in seven minutes without eliminating RNA interference. The detection limit for 5md C was 2. 5 pg per microliter,and the linear range of calibration curve was between 0. 025 to 0. 75 ng / μL with a correlation coefficient greater than 0. 99. The coefficient of variation（ CV） of both inter-day and intra-day were less than 5. 6%. The global DNA methylation levels in the semen of the males with oligoastheno-teratozoospermias were significantly lower than those of control individuals（ P〈0.05）. Conclusion The UPLC-FLD developed in this study should be rapid,convenient,stable and repeatable method for determination of global DNA methylation.