SYBR实时荧光定量PCR检测人乳腺癌细胞AnnexinⅡmRNA方法的建立及应用

摘要

目的：建立检测人AnnexinⅡmRNA水平的SYBR Green实时荧光定量PCR方法，并用其测定人乳腺癌细胞MDA-MB-231和 MCF-7中AnnexinⅡmRNA水平。方法根据人AnnexinⅡ mRNA的保守序列设计特异性引物，提取人乳腺癌细胞总 RNA，并逆转录为cDNA，胶回收纯化PCR产物，并与pGM-T载体连接构建质粒标准品，进一步通过SYBR Green实时荧光定量PCR检测人乳腺癌细胞中 Annexin ⅡmRNA水平。结果该方法标准曲线的r2为0．997，融解曲线分析显示单个峰，pGM-T AnnexinⅡ质粒标准品高、低拷贝数的批内、批间变异系数分别为6．2％、7．8％（高拷贝）和9．1％、12．3％（低拷贝）。进一步研究表明人乳腺癌细胞 MDA-MB-231中 AnnexinⅡmRNA水平显著高于 MCF-7细胞（P＜0．01）。结论建立的检测人 AnnexinⅡmRNA水平的 SYBR Green实时荧光定量PCR方法特异性、重复性好，可用于人乳腺癌细胞中 AnnexinⅡ mRNA的定量检测。%Objective To establish a SYBR Green based real-time fluorescence quantitative PCR method for detecting human Annexin Ⅱ mRNA expression,and to detect the level of Annexin Ⅱ mRNA in human breast cancer cells MCF-7 and MDA-MB-231.Methods The specific primers were designed according to the conserved sequence of human Annexin Ⅱ gene.Total RNAs were extracted from human breast cancer cells(MCF-7,MDA-MB-231),then RNAs were transcribed reversely into cDNAs.The plasmid standards were constructed.The relative expression levels of human Annexin Ⅱ mRNA in human breast cancer cells were detected by this method.Results The square(r2 )of correlation coefficient of the standard curve in this method was 0.997,the melting curve analysis showed the single peak.The the intra-batch and inter-batch variable coefficients in the pGM-T Annexin Ⅱplasmid standard substance were 6.2%,7.8% and 9.1%,12.3% respectively.The further study indicated that AnnexinⅡ mRNA in MDA-MB-231 was higher than that in MCF-7(P<0.01).Conclusion The established SYBR Green real time fluorescence quan-titative PCR method for detecting human AnnexinⅡ is of good specificity and repeatability and can be used for quantitatively detec-ting AnnexinⅡ mRNA in breast cancer cells.