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

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2024-03

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Abstract

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.

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5-Methylcytosine, RNA, Immunoblotting, Staining and Labeling

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https://hdl.handle.net/10161/33824

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https://creativecommons.org/licenses/by-nc/4.0

Published Version (Please cite this version)

10.1016/j.xpro.2023.102815

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Chen, Tingjin, and Hui-Kuan Lin (2024). Protocol to analyze the role of various metabolites in impacting global RNA 5-methylcytosine levels in cultured cells by dot blot. STAR protocols, 5(1). p. 102815. 10.1016/j.xpro.2023.102815 Retrieved from https://hdl.handle.net/10161/33824.

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Lin

Hui-Kuan Lin

Fred and Janet Sanfilippo Distinguished Professor

The research interest in Dr. Lin lab is to understand oncogenic networks between oncogenes and tumor suppressor genes, dissect the regulatory mechanisms underlying  the crosstalk between ageing and cancer, to unravel the role of posttranslational modifications (PTMs) such as ubiquitination  and metabolism in diverse molecular and biological processes important for cancer progression and metastasis, cancer stem regulation, cancer immunity and drug resistance by using biochemical and molecular approaches along with and genetic mouse models, and finally to develop small molecule inhibitors and antibodies targeting critical oncogenic signaling and metabolic vulnerabilities for cancer treatment. His research goals aim to not only reveal fundamental insights and concepts for cancer biology and cancer immunity, but also develop novel paradigms and therapeutic strategies for targeting human cancer and overcoming drug resistance.

Research interests include:

  • Crosstalk between oncogenic and tumor suppressor networks
  • Posttranslational modifications in signaling and cancer
  • Cancer progression and metastasis
  • Biology of normal and cancer stem cells
  • Metabolism in cancer and ageing

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