m6A-ELISA, a simple method for quantifying N6-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 relative m6A levels in mRNA populations from various sources based on an 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 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.

Keywords: ELISA; m6A; mESC; yeast.

FIGURE 1.

Optimization of signal-to-noise for m6A-ELISA. (A) Testing of m6A antibody specificity. Various ratios of in vitro transcribed (IVT) RNA unmodified adenosine (A), and IVT m6A containing RNA (m6A) were used (1%, 0.5%, 0.1%, or 0% m6A IVT RNA). In total, 50 ng IVT RNA was used for each sample. Displayed are the mean and individual OD₄₅₀ values of n = 2 technical replicates from a representative experiment. (B) Primary antibody and blocking reagent testing. Primary antibody (ABClonal-A19841) was incubated with or without 0.5 µg/mL of total RNA to reduce background binding to unmodified RNA. We used 50 ng in vitro transcribed (IVT) RNA generated with unmodified adenosine (A), and 0.01 ng of IVT m6A containing RNA (m6A). Additionally, we used poly(A)-selected mRNA samples from wild-type (WT) (FW1511) diploid cells and cells harboring gene deletion in IME4 (ime4Δ) (FW7030) that were induced to enter meiosis. Displayed are the mean and individual OD₄₅₀ values of n = 2 technical replicates. (C) Similar analysis as in B. We used 50 ng poly(A)-selected mRNA samples from WT and ime4Δ cells that were induced to enter meiosis, and normalized samples to the WT signal. Displayed are n = 4 technical replicates across n = 2 independent experiments (unpaired t-test of P = 0.0004). (D) Primary antibody testing of samples using m6A-ELISA. Two m6A antibodies (Invitrogen-RM362 and Synaptic systems 202003) were tested in the m6A-ELISA. Displayed are the mean and individual OD₄₅₀ values of n = 2 technical replicates from a representative experiment. (E) Similar analysis as in A, except that isolated total RNA was used for the analysis for WT and ime4Δ cells induced to enter meiosis. Displayed are the mean and individual OD₄₅₀ values of n = 2 technical replicates from a representative experiment. (F) Comparison of m6A with a commercial m6A ELISA (EpiQuik). Like in A, the m6A signal for WT and ime4Δ cells were compared. For each experiment, the ime4Δ OD₄₅₀ signals were normalized to WT OD₄₅₀ signal (set to 1). The mean and individual values of n = 4 independent experiments are shown.

FIGURE 2.

Validation of m6A-ELISA. (A) Standards generated from serial dilutions and mixing of IVT RNA with m6A modified adenosine and unmodified adenosine. Each standard dilution contained 50 ng IVT unmodified RNA with different quantities (0–10 pg) of m6A modified IVT RNA. The mean and individual values of n = 3 independent experiments are shown. (B) Quantification of m6A levels using m6A ELISA. mRNA from WT and ime4Δ cells induced to enter meiosis was used for the analysis. A dilution series described in A was used to transform the OD₄₅₀ values to m6A signal (AU) per ng mRNA. The mean and individual values of n = 3 independent experiments are shown. (C) Comparison of m6A ELISA and m6A MS using mRNA from WT and ime4Δ cells. The data from the m6A MS data was obtained from Varier et al. (2022). To make a direct comparison, for each WT vs. ime4Δ experiment the WT signal was set to 1. The mean and individual values of n = 3 independent experiments are shown.

FIGURE 3.

Overview of the m6A-ELISA protocol.

FIGURE 4.

m6A-ELISA detects incremental changes in m6A. (A) Quantification of m6A RNA levels in WT and ime4Δ. WT and ime4Δ samples were mixed in various proportions: 100% vs. 0%, 75% vs. 25%, 50% vs. 50%, 25% vs. 75%, and 0% vs. 100% (WT vs ime4Δ). Signals were normalized to standard curve, and subsequently the WT signal was set to 100%. Unpaired t-test, n = 6 technical replicates in n = 2 independent experiments, each step P < 0.0005. (B) m6A deposition throughout yeast meiosis. Samples were taken at the indicated time points, and m6A levels were determined. Signals were normalized to the standard curve. The mean and individual values of n = 3 replicates are shown. (C) m6A levels determined by m6A-ELISA using mRNA isolated from WT and Mettl3 KO mESC, and Mettl3 KO* mESC line. (*) Also contains a copy of human METTL3 under control of a doxycycline inducible promoter. An amount of 25 ng of RNA was used for the m6A-ELISA. The mean and individual values for the WT and Mettl3 KO n = 4 replicates are shown of two mESC cultures, and Mettl3 KO* n = 3 replicates.

Imke Ensinck