An Improved m6A-ELISA for Quantifying <em>N<sup>6</sup></em>-methyladenosine in Poly(A)-purified mRNAs

<i>N⁶</i> -methyladenosine (m6A) is an abundant internal mRNA modification with roles in regulating cellular and organismal physiology, including development, differentiation, and disease. The deposition of m6A is highly regulated, with various m6A levels across different environmental conditions, cellular states, and cell types. Available methods for measuring bulk m6A levels are often time-consuming, have low throughput, and/or require specialized instrumentation or data analyses. Here, we present a detailed protocol for measuring bulk m6A levels in purified poly(A) RNA samples with m6A-ELISA using a standard-based approach. Critical steps of the protocol are highlighted and optimized, including poly(A) RNA quality controls and antibody specificity testing. The protocol is fast, scalable, adaptable, and cost-effective. It does not require specialized instrumentation, training, or skills in data analysis. We have successfully tested this protocol on mRNAs isolated from budding yeast and mouse cell lines. Key features • <i>N⁶</i> -methyladenosine quantification, including mRNA isolation, can be achieved in two days. • Describes a robust method for poly(A) RNA isolation from total RNA, minimizing non-poly(A) RNA contamination. • Based on Ensinck et al. [1], optimizing poly(A) RNA selection from yeast and mammalian cells and reducing the amount of mammalian mRNA for the assay.