摘要
目的建立实时荧光定量PCR检测皮层肌动蛋白基因表达水平的方法。方法提取人肝癌组织总RNA，RT—PCR扩增CTTN和GAPDH基因片段并分别构建两片段阳性表达载体。采用SYBR Green I实时荧光定量PCR绘制两基因拷贝数标准曲线，实测人肝癌标本并进行方法学评价。结果成功构建pMD18-T（＋）／CTTN和pMD18-T（＋）／GAPDH重组质粒。CTTN和GAPDH基因模板拷贝数分别在1．6×10^3-1．6×10^7和1．6×10^4-1．6×10^7之间时标准曲线有良好线性关系，R^2分别为0．996和0．985。由Ct值统计两基因组内变异系数分别为0．11％～2．25％和0．75％-3．63％；组间变异系数分别为0．83％～1．48％和0．47％～1．36％。组间无统计学差异（P〉0．05）。结论成功建立实时荧光定量PCR检测人肝癌组织CTTN基因的方法，为研究人肝癌组织CTTN基因表达水平奠定了方法学基础。
Objective To develop a real-time fluorescent quantitative polymerase chain reaction （RT-PCR） for the detection of cortactin gene expression in human hepatocellular carcer （HCC） tissue. Methods Total RNA was extracted from human liver cancer tissue samples. Cortactin and GAPDH gene were amplified by RT-PCR and the products of each gene were inserted in a pMD18-T vector to plot standard curves by real-time PCR with SYBR Green I. Five human liver cancer tissue samples were detected based on the standard curves and the threshold cycle （Ct） value was analyzed to evaluate the precision, linearity and reproducibility of this method. Results pMD18-T（＋）/CTrN and pMD18-T（＋）/GAPDH plasmids were successfully constructed. When the concentration of temples was 1.6 × 10^3-1.6 × 10^7 and 1.6 × 10^4-1.6 × 10^7 copies/ix L, prespectively, cortactin and GAPDH standard curves had a good linearity （R^2=0.996 and 0.985）.The repeatability of two genes was 0.11%-2.25% and 0.75%-3.63% （coefficient of variability） respectively within run and 0.83%-1.48% and 0.47%-1.36% （coefficient of variability） respectively between days. No statistical difference was observed between days （P〉0.05）. Conclusion The RT-PCR developed in this study lays a methodological foundation for the study of CTrN gene expression level in human HCC tissue.
出处
《军医进修学院学报》
CAS
2010年第2期171-174,共4页
Academic Journal of Pla Postgraduate Medical School
基金
空军总医院科研课题资助项目 (KZ2007001)
