Dot Blot Assay for Detecting Global N6-Methyladenosine RNA Modification Levels

N⁶-methyladenosine (m⁶A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA) and serves as a key regulator of post-transcriptional gene expression. Methods for investigating m⁶A can be applied at multiple levels of resolution, including global quantification, nucleotide-specific detection, and analysis of particular transcripts of interest. Among these, the dot blot assay provides a straightforward, rapid, and cost-effective approach for semi-quantitative evaluation of global m⁶A modification levels. In this assay, cells are first processed to extract total RNA, which is then diluted and denatured. The RNA samples are spotted onto a nylon membrane, probed with an anti-m⁶A antibody, and visualized by chemiluminescence, with signal intensity indicating the relative abundance of methylation. Finally, quantification and comparison of signal intensities are performed by measuring grayscale values with ImageJ. Compared with sequencing or mass spectrometry-based methods, dot blot requires minimal instrumentation and technical expertise, making it particularly suited for routine screening, preliminary functional studies, and comparative analyses. This protocol provides a detailed and reproducible workflow for implementing the m⁶A dot blot assay in both basic research and translational applications.