Analysis of N6-methyladenosine RNA Modification Levels by Dot Blotting

Abstract

N6-methyladenosine (m6A) 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 m6A modification is also extensively present in non-coding RNAs, including microRNAs (miRNAs), ribosomal RNAs (rRNAs), and transfer RNAs (tRNAs). Common m6A methylation detection techniques play an important role in understanding the biological function and potential mechanism of m6A, mainly including the quantification and specific localization of m6A modification sites. Here, we describe in detail the dot blotting method for detecting m6A 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 m6A level of single RNA species, so as to facilitate further studies of the role of m6A in biological processes.