SYBR Green实时荧光PCR技术在肝细胞癌基因甲基化定量分析中的应用

目的 建立一种简便易行的甲基化定量检测技术,并对肝细胞癌中基因的甲基化情况进行分析,从而对该技术在临床应用中的可行性进行评估.方法 基因组DNA经亚硫酸氢钠修饰处理后,构建包含CDKN2A和ACTB两个基因片段的质粒标准品,通过SYBR Green荧光定量PCR检测并制作标准曲线,并用这一方法对41例肝细胞癌中两种基因进行甲基化定量分析.结果 通过对扩增曲线、熔解曲线、标准曲线以及电泳结果的分析表明,SYBR Green荧光定量PCR对102～108拷贝/μL范围内质粒标准品的扩增具很高特异性、灵敏度和较宽的检测范围;通过对41例肝细胞癌手术标本的检测进一步表明证实了本方法在基因甲基化定量分析中的可行性.结论 SYBR Green荧光定量法作为一种简便易行且具备高通量分析能力的定量检测方法,可以应用于肿瘤中基因甲基化的研究与临床检测.

Detection of methylation in hepatocellular carcinoma using SYBR Green fluorescent quantitative PCR

OBJECTIVE: To establish a quantitative technique for assaying gene methylation in hepatocellular carcinoma (HCC) and evaluate its feasibility for clinical application. METHODS: Following bisulfite modification and PCR amplification, the fragments of CDKN2A and ACTB were cloned into plasmids to generate calibration curves using SYBR Green quantitative PCR, and then these two genes were quantitatively analyzed in 41 cases of HCC specimen. RESULTS: The amplification curve, dissociation curve, calibration curve and electrophoresis analysis showed that SYBR Green fluorescent quantitative PCR could assay 10(2)-10(8) copies/microL of recombinant plasmids with high specificity, high sensitivity and a wide detection range. The tests on 41 cases of HCC specimens further confirmed its feasibility for quantitative analysis of methylation. CONCLUSION: SYBR Green fluorescent PCR is an easy, fast and high-throughout quantitative tool, and it can be used for methylation analysis in basic research or clinical assay.