Protocol to analyze the role of various metabolites in impacting global RNA 5-methylcytosine levels in cultured cells by dot blot.
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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DOI：
10.1016/j.xpro.2023.102815
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发表时间：
2024-03-15
期刊：
影响因子：
--
通讯作者：
Lin, Hui-Kuan
中科院分区：
文献类型：
--
作者：
Chen, Tingjin;Lin, Hui-Kuan
RNA 5-甲基胞嘧啶 (m5C) 修饰对许多生物过程产生严重影响。在这里，我们提供了一个协议，通过斑点印迹分析各种代谢物在影响培养细胞中全局 RNA m5C 水平中的作用。我们描述了用各种代谢物处理培养细胞的步骤； RNA 样品的提取、定量和变性；并进行斑点印迹检测培养细胞中的总体 RNA m5C 水平。然后，我们详细介绍了通过亚甲蓝染色验证输入负载的程序，并使用 ImageJ 进行量化。有关该协议的使用和执行的完整详细信息，请参阅 Chen 等人。根据研究目的用各种代谢物处理培养细胞 从培养细胞中提取、定量和变性 RNA 点印迹检测培养细胞中的总体 RNA 5-甲基胞嘧啶水平 通过亚甲蓝染色验证输入负载并使用 ImageJ 进行定量 出版商注释：进行任何实验方案都需要遵守当地实验室安全和道德机构指南。 RNA 5-甲基胞嘧啶 (m5C) 修饰对许多生物过程产生严重影响。在这里，我们提供了一个协议，通过斑点印迹分析各种代谢物在影响培养细胞中全局 RNA m5C 水平中的作用。我们描述了用各种代谢物处理培养细胞的步骤； RNA 样品的提取、定量和变性；并进行斑点印迹检测培养细胞中的总体 RNA m5C 水平。然后，我们详细介绍了通过亚甲蓝染色验证输入负载的程序，并使用 ImageJ 进行量化。
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. Treatment of cultured cells with various metabolites based on the research purpose Extraction, quantification, and denaturation of RNA from cultured cells Dot blot to detect global RNA 5-methylcytosine levels in cultured cells Verification of input loading by methylene blue staining and quantification using ImageJ Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. RNA 5-methylcytosine (m5C) modification critically impacts many biological processes. Here, we provide a protocol to dissect 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.
有氧糖酵解通过己糖激酶 2 介导的 IγBα 磷酸化促进肿瘤免疫逃避
影响因子：
29
作者：
Guo, Dong;Tong, Yingying;Lu, Zhimin
通讯作者：
Lu, Zhimin
影响因子：
29
作者：
Chen，Tingjin;Xu，Zhi-Gang;Lin，Hui-Kuan
通讯作者：
Lin，Hui-Kuan