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目的通过比较MassARRAY定量分析法与普通亚硫酸盐测序法(BSP)检测心肌梗死大鼠乙醛脱氢酶(ALDH2)基因启动子甲基化的效果,为确定适用于低甲基化基因序列检测的最佳方法提供依据。方法取10只平均体重为(200±10)g的雄性Sprague-Dawley大鼠,将其随机分入对照组和实验组,每组5只。实验组行心脏冠状动脉左前降支结扎术,制备心肌梗死模型。常规饲养7d后,取实验组大鼠心脏梗死边缘区心肌组织,同时在对照组的相同区域取材。提取边缘区心肌组织DNA,分别采用MassARRAY定量分析法和普通BSP检测两组大鼠ALDH2基因核心启动子上游同一段DNA序列甲基化情况。结果 BSP可检测显示目标区域12个CpG位点,但检测结果示对照组和实验组均无DNA甲基化发生;MassARRAY定量分析法可检测显示目标区域10个CpG位点(2号至11号位点),实验组和对照组10个CpG位点均有低度DNA甲基化(<30%),且两组间5号、6号和7号CpG位点甲基化水平的差异均有统计学意义(P值均<0.05)。结论对于低甲基化基因序列,MassARRAY定量分析法较BSP拥有更好的DNA甲基化检测精度和检测效率。
Objective To compare the methylation status of ALDH2 gene promoter in myocardial infarction rats by comparing MassARRAY quantitative analysis with ordinary sulfite sequencing (BSP) Provide the basis for the best method. Methods Ten male Sprague-Dawley rats with an average body weight of (200 ± 10) g were randomly divided into control group and experimental group with 5 rats in each group. The experimental group underwent left anterior descending coronary artery ligation, preparation of myocardial infarction model. After routine feeding for 7 days, the myocardium in the marginal zone of myocardial infarction in the experimental group was taken and the same area of the control group was taken. The DNA of marginal zone myocardium was extracted. The methylation of the DNA sequence of the same segment upstream of the core promoter of ALDH2 gene in two groups of rats was detected by MassARRAY quantitative analysis and ordinary BSP. Results BSP could detect 12 CpG sites in the target area. However, no DNA methylation was detected in the control and experimental groups. MassARRAY quantitative analysis showed that there were 10 CpG sites in the target area (Nos. 2 to 11 Site). All of the 10 CpG sites in the experimental group and the control group had low DNA methylation (<30%), and the methylation levels of CpG sites of No.5, No.6 and No.7 were significantly different between the two groups There was statistical significance (P <0.05). Conclusion MassARRAY quantitative analysis has better DNA methylation detection efficiency and detection efficiency than BSP for hypomethylated gene sequences.