荧光定量RT-PCR检测OATP-E基因表达

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 transporting polypeptide-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/μg RNA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。

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

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 5.47×105copies/μg RNA in MCF-7 cell line and 2.82×104copies/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.