预扩增qPCR方法检测少量小鼠早期胚胎细胞中DNA甲基化相关基因的表达

Detection of expression of methylation-related genes in mouse early embryonic cells using pre-amplification-based real time quantitative PCR

目的:比较3种实时定量PCR(qPCR)方法对少量小鼠早期胚胎细胞中甲基化相关基因表达水平的检测效果,以期获得适合日常检测的方法。方法:分别应用常规qPCR方法、基于等温预扩增的qPCR方法、基于PCR预扩增的qPCR方法3种方案对少量小鼠早期胚胎细胞样本中甲基化相关基因TET1、TET2、TET3、DNMT3A的mRNA表达进行检测。结果:3种方法的灵敏度由高到低依次为基于等温预扩增的qPCR方法、基于PCR预扩增qPCR方法、常规qPCR方法。前两种方法均能在合适的循环数下对少量小鼠胚胎细胞中的多个甲基化相关基因表达进行定量。而基于PCR预扩增的qPCR方法比基于等温预扩增的qPCR方法更经济,适合日常检测。因此,我们选择应用基于PCR预扩增的qPCR方法,检测了小鼠卵细胞以及卵细胞受精后22 h甲基化相关基因表达的变化,结果发现该过程中TET1表达量很低,不易检测到;TET2表达呈降低趋势;TET3和DNMT3A表达显著升高(P<0.01),与文献报遁基本一致。结论:基于等温预扩增的qPCR方法检测少量细胞样品中甲基化相关基因的表达的灵敏度在3种qPCR方法中最高。而基于PCR预扩增的qPCR方法操作简单、灵敏度较高、成本相对低,适合少量细胞样本中多个相关基因表达的实时定量日常检测。

OBJECTIVE：To identify a proper qPCR method for examining expression of methylation-related genes in mouse early embryonic cells.METHEDS：Normal qPCR,isothermal pre-amplification-based qPCR,and PCR pre-amplification-based qPCR methods were applied in quantitative assay of methylation-related genes（TET1,TET2,TET3 and DNMT3A） in mouse early embryonic cells.RESULTS：The sensitivity of these three methods decreased from isothermal pre-amplification-based qPCR method,PCR pre-amplification-based qPCR method to normal qPCR method.The former two methods detected expression of mediylation-related genes in a few mouse embryonic cells within a proper PCR cycle number but the PCR pre-amplification-based qPCR method has lower cost than isothermal pre-amplification-based qPCR method.Therefore,we used PCR pre-amplification-based qPCR method to examine methylation-related genes in mouse oocytes and early embryonic cells after 22 h fertilization.The results showed that the expression level of TET1 mRNA was very low and not detectable,the expression of TET2 decreased,and the expression of TET3 and DNMT3 A significantly increased（P〈0.01） in this process,which was consistent with reported results.CONCLUSION：The sensitivity of isothermal pre-amplification-based qPCR method was the highest among the three qPCR methods.However,the PCR pre-amplification-based qPCR method is the most suitable one for examining multigene expression in a few cells in daily practice due to the simple procedure,relatively high sensitivity and low expenditure.