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Development and Application of Detection Methods for Capture and Transcription Elongation Rate of Bacterial Nascent RNA

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生物化学与生物物理进展 2024年第9期51卷 2249-2260页

作者： LI Yuan-Yuan WANG Yu-Ting WU Zi-Chun LI Hao-Xuan FEI Ming-Yue SUN Dong-Chang gualerzi o.claudio FABBRETTI Attilio GIULIoDoRI Anna Maria MA Hong-Xia HE Cheng-Guang Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development College of Life SciencesJilin Agricultural UniversityChangchun 130118China School of Biosciences and Veterinary Medicine University of CamerinoCamerino(MC)62032Italy College of Biotechnology and Bioengineering Zhejiang University of TechnologyHangzhou 310014China

objective Detection and quantification of RNA synthesis in cells is a widely used technique for monitoring cell viability,health,and metabolic *** exposure to environmental stimuli,both the internal reference gene and target gene would be *** a result,it is imperative to consider the accurate capture of nascent RNA and the detection of transcriptional levels of RNA following environmental *** study aims to create a Click Chemistry method that utilizes its property to capture nascent RNA from total RNA that was stimulated by the *** 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 *** 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 *** 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 *** 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.

关键词： nascent RNA selection Click Chemistry fluorescence molecular beacon

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