Development and Application of Detection Methods for Capture and Transcription Elongation Rate of Bacterial Nascent RNA
10.16476/j.pibb.2023.0478
- VernacularTitle:细菌新生RNA捕获及转录延伸速率检测方法的开发与应用
- Author:
Yuan-Yuan LI
1
;
Yu-Ting WANG
1
;
Zi-Chun WU
1
;
Hao-Xuan LI
1
;
Ming-Yue FEI
2
;
Dong-Chang SUN
2
;
O. Claudio GUALERZI
3
;
Attilio FABBRETTI
3
;
Anna Maria GIULIODORI
3
;
Hong-Xia MA
1
;
Cheng-Guang HE
1
Author Information
1. Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development, College of Life Sciences, Jilin Agricultural University, Changchun 130118, China
2. College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China
3. School of Biosciences and Veterinary Medicine, University of Camerino, Camerino (MC) 62032, Italy
- Publication Type:Journal Article
- Keywords:
nascent RNA selection;
Click Chemistry;
fluorescence molecular beacon
- From:
Progress in Biochemistry and Biophysics
2024;51(9):2249-2260
- CountryChina
- Language:English
-
Abstract:
ObjectiveDetection 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. MethodsThe 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). ResultsThe 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. ConclusionCompared 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.
