实时荧光定量检测人血浆中RASSF1A基因启动子甲基化

Plasma RASSF1A gene methylation quantification by real-time MSP

目的建立检测人血浆DNA中RASSF1A基因甲基化的方法。方法以肺癌细胞株NCI-H460作为RASSF1A基因启动子区甲基化阳性样本,传统酚/氯仿法提取细胞DNA,经紫外分光光度计定量后以10倍稀释的浓度梯度依次投入到200μl健康人血浆中,制备得到模拟肺癌患者血浆。用磁珠法从模拟血浆样本和15例早期肺癌患者血浆中提取总DNA。对血浆DNA模板进行亚硫酸氢盐化学修饰,并以TaqMan技术的实时荧光定量甲基化特异性PCR(MSP)进行检测。以模拟血浆样品作为标准品,用外标准曲线法对肺癌患者血浆中的甲基化RASSF1A进行定量。结果实时荧光定量MSP对模拟肺癌患者血浆的检测最低限为150拷贝(甲基化的肿瘤DNA)/ml(血浆)。15例肺癌患者5例RASSF1A甲基化阳性(33.3%)。5例肺癌患者血浆中甲基化的RASSF1A基因的浓度分别为2.56×103,8.20×104,1.45×104,3.31×104和4.24×104拷贝/ml。结论实时荧光定量MSP法检测肺癌患者血浆DNA中RASSF1A基因甲基化,灵敏度高,特异性强。

Objective To develop a quantitative method for the quantification of RASSF1A gene methylation in plasma by real-time fluorescent quantitative MSP. Methods After purification by phenol-chloroform method and quantification by spectrophotometrie measurement, genomic DNA with methylated RASSF1A gene in lung cancer cell line NCI-H460 was added into plasma samples of healthy volunteers with serial dilution. Circulating DNA in simulated plasma samples and specimen of 15 patients with early lung cancer was extracted by magnetic bead protocol and modified by bisulfite. The DNA concentrations of cell-free methylated RASSF1A in the plasma samples of lung cancer patients were quantified using external reference method with standard curve eonstructed by simulated plasma. Results Real-time MSP could detect 150 copies of methylated RASSF1A DNA in one milliliter of simulated plasma. Methylated RASSF1A DNA was found in 33.3% （5/15） of early lung cancer patients. The concentration of methylated RASSF1A in plasma of five patients with early lung cancer was 2.56×10^3 ,8.20×10^4 ,1.45×10^4 ,3.31×10^4 and 4.24×10^4 copies/ml respectively. Conclusions The real-time MSP assay allows quantitative detection of RASSF1A gene hypermethylation in plasma with high sensivity and specificity. It might be a useful tool for diagnosis of early lung cancer.