Establishment and application of SYBR real time fluorescence quantitative PCR for detecting Annexin Ⅱ mRNA of human breast cancer cells / 重庆医学

ABSTRACT

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.