乳腺癌组织中ERα mRNA和PR mRNA检测方法的建立和应用

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。

The establishment of ERa and PR mRNA assays and their application in breast cancer

To establish a specific fluorogenic quantitative method for detecting the oestrogen receptor-alpha(ERa) and progesterone receptor(PR) gene,and to explore their expression both in benign and cancer tissues,based on fluorescent Taq Man methodology,a real-time quantitative RT-PCR was set up.In this method,cloning vector pMD18-ERa and pMD18-PR were constructed as standard plasmids.RNA quantification was based on the threshold cycle(Ct) values when using GeneAmp 7500 Sequence Detection Systems to examine the specific expression of ERa and PR in 38 cases with breast cancer and 32 cases with benign tumor(the controls).The detection linear range of the assay was 10~3 ~ 10~8 copy/jug RNA.The intra- and inter-assay coefficient of variation of the ERa and PR for low value and high value was 5.07%and 11.28%respectively.The ERαmRNA copy number was 6.76×10~5 copy/jug RNA(4.17×10~5,9.34×10~5) in breast cancers and 1.54×10~5 copies/μg RNA(1.02×10~5 2.06×10~5) in controls.The PR mRNA copy number was 1.02×10~6 copy/μg RNA(6.81×10~5,1.36×10~6) in cancer and 4.93×10~5 copy/jug RNA(3.21×10~5,6.65×10~5) in controls,respectively.ERαmRNA levels were significantly elevated in breast cancer tissues when compared to control tissues(P<0.05),and were well associated with the clinicopathological features of breast cancer.PR mRNA did not reveal significant difference between cancers and controls(p>0.05).Thus,a real-time quantitative RT-PCR method for detecting the expression of ERa and PR gene in the breast tissue has been successfully established., ERαand PR detection is a very important clinical test in breast cancer,which might not only be an important prognostic and predictive marker but also be used to determine optimal treatment strategies.