m6A-ELISA, a simple method for quantifyingN6-methyladenosine from mRNA populations

Abstract

N6-methyladenosine (m6A) is a widely studied and abundant RNA modification. The m6A mark regulates the fate of RNAs in various ways, which in turn, drives changes in cell physiology, development, and disease pathology. Over the last decade, numerous methods have been developed to map and quantify m6A sites genome-wide through deep sequencing. Alternatively, m6A levels can be quantified from a population of RNAs using techniques such as liquid chromatography-mass spectrometry or thin layer chromatography. However, many methods for quantifying m6A levels involve extensive protocols and specialized data analysis, and often only a few samples can be handled in a single experiment. Here, we developed a simple method for determining m6A levels in mRNA populations from various sources based on enzyme-linked immunosorbent-based assay (m6A-ELISA). We have optimized various steps of m6A-ELISA such as sample preparation and the background signal resulting from the primary antibody. We validated the method using mRNA populations from budding yeast and mouse embryonic stem cells. The full protocol takes less than a day, requiring only 25 ng of mRNA. The m6A-ELISA protocol is therefore quick, cost-effective, and scalable, making it a valuable tool for determining relative m6A levels in samples from various sources that could be adapted to detect other mRNA modifications.

The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles

“…To confirm these previous findings, we determined m6A levels in diploid cells undergoing early meiosis (4 hours in sporulation medium (SPO)) harbouring gene deletion in MUM2 and IME4 ( mum2 Δ and ime4 Δ). Both deletion mutants showed a severe reduction in m6A over A levels as determined by LC-MS (Figure 1D), consistent with previous measurements (Ensinck et al, 2022). We also quantified m6A levels in a SLZ1 deletion strain.…”

“…RNA was extracted, and polyA RNA was purified. m6A levels were determined by a non-commercial m6A-ELISA assay (Ensinck et al, 2022). The means of n=3 biological replicates are shown of WT and ime4 Δ, and n=6 for dyn2 Δ.…”

“…For ELISA we either used the EpiQuik m6A RNA methylation quantification kit from EpiGentek (P-9005) according to manufacturer’s protocol or an m6A ELISA protocol previous described (Ensinck et al, 2022).…”

N6-methyladenosine (m6A) reader Pho92 is recruited co-transcriptionally and couples translation to mRNA decay to promote meiotic fitness in yeast

“…For ELISA, we either used the EpiQuik m6A RNA methylation quantification kit from EpiGentek (P-9005) according to manufacturer’s protocol or the protocol we developed ( Ensinck et al, 2022 ). In our method 90 µl binding solution (ab156917) and 50 ng of twice polyA selected RNA (similar to LC-MS/MS above) were added to each well of a 96-well plate (ab210903).…”