摘要
Studying the expression level of mRNA in living cells will offer tremendous opportunities for ad-vancement in cell biology research,disease diagnostics,and drug discovery.In this paper,a molecular beacon(MB)specific for the important tumor suppressor gene p21 has been designed and synthesized.The fluorescence signal was detected in real-time after the MB entered the cytoplasm of nasopharyn-geal carcinoma cells.After injecting the p21MB into nasopharyngeal carcinoma cell and p33-trans-fected nasopharyngeal carcinoma cell,the consistent increase of fluorescent signal intensity was de-tected in both cell lines,and maximum fluorescence intensity achieved in about 15 min.In about 4 min following microinjection,the fluorescence increasing rate was significantly different between these two cell lines,which indicate the different p21 mRNA expression levels.The results obtained in the real-time detection were also validated by RT-PCR.Analysis of the initial fluorescence increasing rate can effi-ciently reduce the side effect of enzyme and improve the accuracy in living cell mRNA detection.
Studying the expression level of mRNA in living cells will offer tremendous opportunities for advancement in cell biology research, disease diagnostics, and drug discovery. In this paper, a molecular beacon （MB） specific for the important tumor suppressor gene p21 has been designed and synthesized. The fluorescence signal was detected in real-time after the MB entered the cytoplasm of nasopharyngeal carcinoma cells. After injecting the p21MB into nasopharyngeal carcinoma cell and p33-transfected nasopharyngeal carcinoma cell, the consistent increase of fluorescent signal intensity was detected in both cell lines, and maximum fluorescence intensity achieved in about 15 min. In about 4 min following microinjection, the fluorescence increasing rate was significantly different between these two cell lines, which indicate the different p21 mRNA expression levels. The results obtained in the real-time detection were also validated by RT-PCR. Analysis of the initial fluorescence increasing rate can efficiently reduce the side effect of enzyme and improve the accuracy in living cell mRNA detection.
基金
the National Key Basic Research Program(Grant No.2002CB513110)
the Key Technologies Research Development Program of China(Grant Nos.2003BA310A16and2005EP090026)
International Technologies Collaboration Program of China(Grant No.2003DF000039)
the National Natural Science Foun-dation of China(Grant Nos.90606003and20475015)
Key Project of Hunan Province Technology Plan of China(Grant No.0399Y1006)
关键词
P21
mRNA
鼻咽癌
癌细腻
荧光增强率
基因表达
molecular beacon, p21 mRNA, nasopharyngeal carcinoma cell, living cell imaging, fluorescence increasing rate
