荧光定量RT-PCR检测OATP-E基因表达

Quantification of organic anion transporting polypeptide-E gene expression using the fluorogenic quantitative RT-PCR method

目的:建立荧光定量RT-PCR检测肿瘤细胞有机阴离子转运多肽E(organic anion transportingpolypeptide-E,OATP-E)mRNA的方法,了解乳腺癌细胞株MCF-7和对阿霉素耐药的乳腺癌细胞株MCF-7/ADR中OATP-E mRNA的表达水平。方法:构建重组质粒,绘制荧光定量RT-PCR检测OATP-E mRNA的标准曲线,并运用此方法检测MCF-7/ADR和MCF-7细胞株中OATP-E mRNA的表达。结果:荧光定量RT-PCR检测MCF-7和MCF-7/ADR细胞OATP-E的基因表达,重复10次实验所得结果平均分别为5.47×105拷贝数/μgRNA和2.82×104拷贝数/μg RNA,两者相差约19.4倍,P<0.05。结论:成功建立了荧光定量RT-PCR检测OATP-E基因表达的方法,检测结果用绝对拷贝数表示,定量准确可靠,并有利于标准的统一。初步应用发现乳腺癌耐药株MCF-7/ADR中OATP-E mRNA的表达量比不耐药株MCF-7减少。

Objective： To establish a fluorogenic quantitative RT-PCR method for detecting the expression of OATP-E gene in tumor cells, and to investigate the expression of organic anion transporting polypeptide-E （OATP-E） gene in breast cancer cell lines MCF-7 and MCF-7/ADR. Method： Fluorogenic quantitative RT-PCR （FQ- RT- PCR） was set up to examine the specific expression of OATP-E gene in MCF-7 and MCF-7/ADR cell lines, Results： After 10 duplicate tests with the FQ-RT-PCR method, the average level of the OATP-E mRNA was 5.47 × 10^5 copies/μg RNA in MCF-7 cell line and 2.82 × 10^4copies/μg RNA in MCF-7/ADR cell line, respectively, therefore the former was 19,4 times greater than the latter. Conclusions： Using FQ-RT-PCR, we can get an accurate quantitative result of OATP-E gene expression . Moreover, OATP-E gene expression in the drug resistant breast cancer cell line MCF-7/ADR is at a lower level than that in cell line MCF-7.