Objective Detection and quantification of RNA synthesis in cells is a widely used technique for monitoring cell viability,health,and metabolic rate.After exposure to environmental stimuli,both the internal reference g...Objective Detection and quantification of RNA synthesis in cells is a widely used technique for monitoring cell viability,health,and metabolic rate.After exposure to environmental stimuli,both the internal reference gene and target gene would be degraded.As a result,it is imperative to consider the accurate capture of nascent RNA and the detection of transcriptional levels of RNA following environmental stimulation.This study aims to create a Click Chemistry method that utilizes its property to capture nascent RNA from total RNA that was stimulated by the environment.Methods The new RNA was labeled with 5-ethyluridine(5-EU)instead of uracil,and the azido-biotin medium ligand was connected to the magnetic sphere using a combination of“Click Chemistry”and magnetic bead screening.Then the new RNA was captured and the transcription rate of 16S rRNA was detected by fluorescence molecular beacon(M.B.)and quantitative reverse transcription PCR(qRT-PCR).Results The bacterial nascent RNA captured by“Click Chemistry”screening can be used as a reverse transcription template to form cDNA.Combined with the fluorescent molecular beacon M.B.1,the synthesis rate of rRNA at 37℃is 1.2 times higher than that at 15℃.The 16S rRNA gene and cspI gene can be detected by fluorescent quantitative PCR,it was found that the measured relative gene expression changes were significantly enhanced at 25℃and 16℃when analyzed with nascent RNA rather than total RNA,enabling accurate detection of RNA transcription rates.Conclusion Compared to other article reported experimental methods that utilize screening magnetic columns,the technical scheme employed in this study is more suitable for bacteria,and the operation steps are simple and easy to implement,making it an effective RNA capture method for researchers.展开更多
Stimuli-responsive polymer gels have recently attracted great attention due to their heat/solvent resistance,dimensional stability,and unique sensitivity to external stimuli.In this work,we synthesized thiol-functiona...Stimuli-responsive polymer gels have recently attracted great attention due to their heat/solvent resistance,dimensional stability,and unique sensitivity to external stimuli.In this work,we synthesized thiol-functionalized tetraphenylethylene(TPE)and constructed polymer gels through thiol-ene click reaction.The synthetic process of the polymer gels could be monitored by fluorescence emission of TPE moieties based on aggregation-induced emission mechanism.In addition,due to the dual redox-and acid responsiveness of the polymer gels,in the presence of dithiothreitol and trifluoroacetic acid,fluorescence quenching of the polymer gels can be observed.This stimuli-responsive characteristics endows the polymer gels with potential applications in fluorescent sensing and imaging,cancer diagnosis and selfhealing materials.展开更多
文摘Objective Detection and quantification of RNA synthesis in cells is a widely used technique for monitoring cell viability,health,and metabolic rate.After exposure to environmental stimuli,both the internal reference gene and target gene would be degraded.As a result,it is imperative to consider the accurate capture of nascent RNA and the detection of transcriptional levels of RNA following environmental stimulation.This study aims to create a Click Chemistry method that utilizes its property to capture nascent RNA from total RNA that was stimulated by the environment.Methods The new RNA was labeled with 5-ethyluridine(5-EU)instead of uracil,and the azido-biotin medium ligand was connected to the magnetic sphere using a combination of“Click Chemistry”and magnetic bead screening.Then the new RNA was captured and the transcription rate of 16S rRNA was detected by fluorescence molecular beacon(M.B.)and quantitative reverse transcription PCR(qRT-PCR).Results The bacterial nascent RNA captured by“Click Chemistry”screening can be used as a reverse transcription template to form cDNA.Combined with the fluorescent molecular beacon M.B.1,the synthesis rate of rRNA at 37℃is 1.2 times higher than that at 15℃.The 16S rRNA gene and cspI gene can be detected by fluorescent quantitative PCR,it was found that the measured relative gene expression changes were significantly enhanced at 25℃and 16℃when analyzed with nascent RNA rather than total RNA,enabling accurate detection of RNA transcription rates.Conclusion Compared to other article reported experimental methods that utilize screening magnetic columns,the technical scheme employed in this study is more suitable for bacteria,and the operation steps are simple and easy to implement,making it an effective RNA capture method for researchers.
基金supported by the National Natural Science Foundation of China (No.51773190 and No.51973206)。
文摘Stimuli-responsive polymer gels have recently attracted great attention due to their heat/solvent resistance,dimensional stability,and unique sensitivity to external stimuli.In this work,we synthesized thiol-functionalized tetraphenylethylene(TPE)and constructed polymer gels through thiol-ene click reaction.The synthetic process of the polymer gels could be monitored by fluorescence emission of TPE moieties based on aggregation-induced emission mechanism.In addition,due to the dual redox-and acid responsiveness of the polymer gels,in the presence of dithiothreitol and trifluoroacetic acid,fluorescence quenching of the polymer gels can be observed.This stimuli-responsive characteristics endows the polymer gels with potential applications in fluorescent sensing and imaging,cancer diagnosis and selfhealing materials.
