Analysis of N ⁶ -methyladenosine RNA Modification Levels by Dot Blotting

N ⁶ -methyladenosine (m ⁶ A) is the most prevalent internal modification of eukaryotic messenger RNAs (mRNAs), affecting their fold, stability, degradation, and cellular interaction(s) and implicating them in processes such as splicing, translation, export, and decay. The m ⁶ A modification is also extensively present in non-coding RNAs, including microRNAs (miRNAs), ribosomal RNAs (rRNAs), and transfer RNAs (tRNAs). Common m ⁶ A methylation detection techniques play an important role in understanding the biological function and potential mechanism of m ⁶ A, mainly including the quantification and specific localization of m ⁶ A modification sites. Here, we describe in detail the dot blotting method for detecting m ⁶ A levels in RNA (mRNA as an example), including total RNA extraction, mRNA purification, dot blotting, and data analysis. This protocol can also be used to enrich specific RNAs (such as tRNA, rRNA, or miRNA) by isolation technology to detect the m ⁶ A level of single RNA species, so as to facilitate further studies of the role of m ⁶ A in biological processes. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.75231.