肝癌实时定量RT-PCR中适宜内参基因的选择

Selection of optimal internal reference genes in human hepatocellular carcinoma using real-time quantitative RT-PCR

目的探讨肝癌（HCC）组织及细胞系实时定量RT-PCR最佳的内参基因选择。方法通过实时定量RT-PCR,检测HMBS、CTBP1、HPRT1、UBC、B2M、SDHA、RPLI3A、EIF4A1、TUBB、YWHAZ、ACTB、GUSB、GAPDH、18 s rRNA和28 s rRNA 15个内参基因在70例HCC、癌旁肝组织及阿法替尼作用后4个HCC细胞系的表达情况。并使用NormFinder及geNorm软件分析最优化的内参基因。结果 15个内参基因表达存在差异,Cq值在8~29.2间。GAPDH在HCC及癌旁肝组织表达差异有显著性（P〈0.05）。结论NormFinder及geNorm软件均显示HMBS＋UBC组合为肝组织的最佳内参,HMBS＋RPLI3A组合为体外肝癌细胞系的最适宜内参。

【Objective】 To investigate the selection of optimal internal reference genes in human hepatocellular carcinoma（HCC） using real-time quantitative RT-PCR.【Methods】 Expression of 15 genes（HMBS,CTBP1,HPRT1,UBC,B2M,SDHA,RPLI3A,EIF4A1,TUBB,YWHAZ,ACTB,GUSB,GAPDH,18s rRNA and 28s rRNA） was detected with real-time quantitative RT-PCR in 70 cases of HCC and their adjacent non-cancerous liver tissues,also in 4 HCC cell lines treated with afatinib.The optimal internal genes were selected by NormFinder and GeNorm softwares.【Results】 The genes studied displayed a wide expression range with Cq values between 8 and 29.2 in both liver tissues and HCC cell lines.GAPDH showed significant difference in expression between malignant and nonmalignant pairs.【Conclusions】 NormFinder and GeNorm both indicated that the optimal internal gene for liver tissue was the combination of HMBS and UBC,whereas the combination of HMBS and RPLI3A was the most stable reference gene for in vitro experiments with HCC cell lines.