摘要
目的建立一种实时荧光RT-PCR定量检测乳腺组织中BRMS1mRNA表达水平的方法。方法采明逆转录聚合酶链反应(RT-PCR)扩增目的基因片段,并实时检测产物的荧光强度,根据标准品建立的标准曲线,计算出待测样本中BRMS1mRNA含量,并以BRMS1mRNA与GAPDHmRNA的比值作为BRMS1mRNA的表达水平。结果实时荧光RT-PCR检测BRMS1及GAPDH的线性范围为4×102-4×108拷贝/μl。批内和批间变异系数分别为3.9%-4.6%和4.8%-5.5%。BRMS1mRNA表达范围为0-1.65.结论实时荧光定量检测BRMS1mRNA含量的方法具有较高的灵敏度和较好的重复性。
Objective To establish a real time fluorescence RT-PCR for quantitative determination of the expression level of BRMSImRNA in tissue of breast cancer. Methods The fluorescence of the PCR product was detected continuously during amplification. According to the standard curve created by standard, the expression level of BRMS1 were determined and the results were presented as the ratios of BRMSlmRNA to GAPDHmRNA .Results The detection range of the assay was from 4×10^2-4×10^8 copies/μl.The coefficient of variation values of both intra-experimental and inter-experimental were 3.9%-4.6% and 4.8%-5.5% respectively. The range of BRMS1 expression is from 0 -1.65 . Conclusions The method of quantitative BRMS1 by read time fluorescent RT-PCR has high sensitivity and reproducibility.
