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建立测定雌激素受体α(ERα)mRNA和孕激素受体(PR)mRNA的实时荧光定量RT-PCR,探讨两者在乳腺癌组织及良性乳腺肿瘤组织中的表达水平。分别以pMD18 ERα和pMD18-PR质粒为定量模板,用循环阈值(Ct)定量起始模板,在荧光TaqMan方法的基础上建立了测定ERαmRNA和PR mRNA的实时荧光定量RT-PCR,并分别测定48例乳腺癌组织及28例良性乳腺肿瘤组织中ERαmRNA和PR mRNA的表达水平。ERαmRNA和PR mRNA测定的线性范围为10~3～10~8copy/μg RNA;ERαmRNA和PR mRNA高值和低值的批内和批间变异系数(CV)在5.07%～11.28%之间。ER mRNA在乳腺癌组织中的表达量为6.76×10~5copy/μg RNA(4.17×10~5,9.34×10~5),良性乳腺肿瘤组织中的含量为1.54×10~5 copy/μg RNA(1.02×10~5,2.06×10~5),乳腺癌组织的表达量高于良性组织(P<0.05);PR mRNA在乳腺癌组织中的表达量为1.02×10~6copy/μg RNA(6.81×10~5,1.36×10~6),良性乳腺肿瘤组织中的含量为4.93×10~5 copy/μg RNA(3.21×10~5,6.65×10~5),两者表达无明显差异(P>0.05)。ERαmRNA和PR mRNA在乳腺癌和良性乳腺肿瘤组织中的表达存在相关性。我们建立的测定ERαmRNA和PR mRNA的实时荧光定量RT PCR灵敏、稳定、重复性好,可供临床检测和研究。ERαmRNA和PR mRNA基因表达水平可作为预测治疗效果及判断预后的重要指标。
To establish a real-time fluorescence quantitative RT-PCR assay to determine the mRNA levels of estrogen receptor alpha (ERα) and progesterone receptor (PR) mRNA in breast cancer and benign breast tumors. The real-time fluorescent quantitative RT-PCR for the determination of ERαmRNA and PR mRNA was established based on the TaqMan assay using the pMD18 ERα and pMD18-PR plasmids as the template and the initial threshold value (Ct) Cases of breast cancer and 28 cases of benign breast tumor tissues ERαmRNA and PR mRNA expression levels. The linear range of ERα mRNA and PR mRNA was 10 ~ 3 ~ 10 ~ 8copy / μg RNA. The intra-assay and inter-assay CVs of ERαmRNA and PR mRNA ranged from 5.07% to 11.28%. The expression of ER mRNA in breast cancer tissues was 6.76 × 10 ~ 5copy / μg RNA (4.17 × 10 ~ 5,9.34 × 10 ~ 5), and that in benign breast tumors was 1.54 × 10 ~ 5 copies / μg RNA (1.02 × 10 ~ 5,2.06 × 10 ~ 5), the expression of breast cancer was higher than that of benign tissues (P <0.05). The expression of PR mRNA in breast cancer tissues was 1.02 × 10 ~ 6copy / μg RNA 6.81 × 10 ~ 5 and 1.36 × 10 ~ 6). The content of benign breast tumor was 4.93 × 10 ~ 5 copies / μg RNA (3.21 × 10 ~ 5,6.65 × 10 ~ 5) (P> 0.05). There is a correlation between the expression of ERαmRNA and PR mRNA in breast cancer and benign breast tumor. We established real-time fluorescence quantitative RT-PCR for the determination of ERα mRNA and PR mRNA, which is sensitive, stable and reproducible for clinical testing and research. ERαmRNA and PR mRNA gene expression levels can be used as an important indicator of predicting the therapeutic effect and prognosis.