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 f...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.展开更多
Advanced fluorescence microscopy including single-molecule localization-based super-resolution imaging techniques requires bright and photostable dyes orproteins asfluorophores.The photophysical properties of fluoroph...Advanced fluorescence microscopy including single-molecule localization-based super-resolution imaging techniques requires bright and photostable dyes orproteins asfluorophores.The photophysical properties of fluorophores have been proven to be crucial for super-resolution microscopy's localization precision and imaging resolution.Fluorophores TAMRA and Atto Rho6 G,which can interact with macrocyclic host cucurbit[7]uril(CB7) to form host-guest compounds,were found to improve the fluorescence intensity and lifetimes of these dyes.We enhanced the localization precision of direct stochastic optical reconstruction microscopy(dSTORM) by introducing CB7 into the imaging buffer,and showed that the number of photons as well as localizations of both TAMRA and Atto Rho6 G increase over 2 times.展开更多
基金the National Key Basic Research Program(Grant No.2002CB513110)the Key Technologies Research Development Program of China(Grant Nos.2003BA310A16and2005EP090026)+2 种基金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)
文摘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.
基金supported by the National Natural Science Foundation of China(31330082,21373200,21525314)the Instrument Developing Project of the Chinese Academy of Sciences(YZ201455)
文摘Advanced fluorescence microscopy including single-molecule localization-based super-resolution imaging techniques requires bright and photostable dyes orproteins asfluorophores.The photophysical properties of fluorophores have been proven to be crucial for super-resolution microscopy's localization precision and imaging resolution.Fluorophores TAMRA and Atto Rho6 G,which can interact with macrocyclic host cucurbit[7]uril(CB7) to form host-guest compounds,were found to improve the fluorescence intensity and lifetimes of these dyes.We enhanced the localization precision of direct stochastic optical reconstruction microscopy(dSTORM) by introducing CB7 into the imaging buffer,and showed that the number of photons as well as localizations of both TAMRA and Atto Rho6 G increase over 2 times.
