mdr-1基因表达的荧光定量RT-PCR法检测  

Detection of multidrug-resistance mdr-1 gene expression using real-time RT-PCR

作　　者：孔庆暖[1] 郭成浩[2] 

机构地区：[1]青岛市市立医院病理科,山东青岛266011 [2]山东大学医学院病理学教研室,山东济南250012

出　　处：《中华肿瘤防治杂志》2007年第3期202-204,219,共4页Chinese Journal of Cancer Prevention and Treatment

摘　　要：目的:建立用实时荧光定量RT-PCR法在mRNA水平上检测肿瘤细胞中多药耐药基因(mdr-1基因)表达的方法。方法:利用RT-PCR方法从耐药肿瘤细胞MCF-7/ADR中扩增出目的基因片段,用TA克隆的方法将基因片段插入到PGEM-T质粒载体中,在Line-gene荧光定量检测系统上建立检测mdr-1基因表达的标准曲线。结果:成功构建了mdr-1基因的质粒重组子,建立了在mRNA水平定量检测mdr-1基因的标准曲线,相关系数为0.997,可稳定检测出40μL体系中102拷贝数的模板。重复5次实验组内和组间变异系数均<1.0%。结论:用基因克隆法可快速准确地构建mdr-1基因的标准曲线,定量检测mdr-1基因的表达,简单易行,重现性好。OBJECTIVE： To est--ablish a method for detection of the expression of mdr-1 gene in tumor cells via real-time RT-PCR. METHODS： The mdr-1 PCR product was subcloned into PGEM-T vector to contrust recombinant plasmid. After optimizing the thermcycling conditions, a quantitative RT PCR assay to quantify mdr-1 gene expression was successfully developed on Line-gene real-time PCR detection system. RESULTS： The standard curve was successfully established and the linear relationship in statistics was good with the regression value 0. 997. The detection limit of the real time PCR assay was 102 copies of template in a 40 μL reaction volume. The intra-assay and interassay coefficients of variation were smaller than 1.0^2. CONCLUSION： The method of detection of the expression of mdr-1 gene using standard curve constructed by TA cloning is easily performed and time-consuming, and has good reproducibility and reliability.

关 键 词：乳腺肿瘤/遗传学 肿瘤细胞 培养的 基因 MDR 基因表达 逆转录聚合酶链反应 荧光 

分 类 号：R73-3[医药卫生—肿瘤]