Protocol to analyze the role of various metabolites in impacting global RNA 5-methylcytosine levels in cultured cells by dot blot

Autor: Tingjin Chen, Hui-Kuan Lin
Jazyk: angličtina
Rok vydání: 2024
Předmět:
Zdroj: STAR Protocols, Vol 5, Iss 1, Pp 102815- (2024)
Druh dokumentu: article
ISSN: 2666-1667
DOI: 10.1016/j.xpro.2023.102815
Popis: Summary: RNA 5-methylcytosine (m5C) modification critically impacts many biological processes. Here, we provide a protocol to analyze the role of various metabolites in impacting global RNA m5C levels in cultured cells by dot blot. We describe steps for treating cultured cells with various metabolites; extracting, quantifying, and denaturing RNA samples; and performing dot blot to detect global RNA m5C levels in cultured cells. We then detail procedures to verify the input loading by methylene blue staining and quantify using ImageJ.For complete details on the use and execution of this protocol, please refer to Chen et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
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