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

Summary

Here, we present a protocol to semi-quantitatively assess global m⁶A levels using dot blot. Total RNA is extracted, denatured, spotted on a nylon membrane, probed with anti-m⁶A antibody, and visualized by chemiluminescence. Signal intensity, quantified by ImageJ grayscale analysis, reflects relative methylation abundance, providing a reproducible workflow for research.

Abstract

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.

Introduction

N⁶-methyladenosine (m⁶A) is the most prevalent internal modification in eukaryotic messenger RNA (mRNA) and plays an essential role in post-transcriptional regulation, including RNA stability, splicing, degradation, translation, and export^1,2,3. Dysregulation of m⁶A modification has been implicated in diverse physiological and pathological processes, including tumor initiation, progression, and metastasis^4,5. In osteosarcoma, elevated m⁶A levels have been observed in both cell lines and ti....

Protocol

NOTE: The protocol is organized into three sections: RNA preparation, dot blot assay, and quantification. Table of Materials summarizes the required materials, Supplementary Table 1 details the reagent formulations, and Figure 1 illustrates the experimental workflow.

1. RNA preparation

1. Cell preparation
   1. Take out 143B cells with stable SNHG21-knockdown (shSNHG21), negative control cells (shCtrl), and cells treated with 3-deazaadenosine (3-DAA; [100 µM, 24 h]) and meclofenamic acid (MA; [100 µM, 24 h]) from the 37 °C incubato....

Results

To rigorously validate the performance of the dot blot assay and to examine the role of SNHG21 in regulating global m⁶ A levels, 143B cells were subjected to pharmacological perturbations that either suppress or enhance m⁶ A methylation and monitored m⁶ A abundance in both directions.

Dot blot quantification of global m⁶ A abundance
We first evaluated the ability of this assay to detect reductions in m⁶ A levels by inhibiting the m

Discussion

In this study, knockdown of SNHG21 in osteosarcoma 143B cells resulted in reduced m⁶A signal intensity, demonstrating the value of this assay for evaluating whether genetic modifications affect RNA methylation. Additionally, this assay serves as a tool for multiple applications, including: 1) Functional studies and mechanistic validation: This assay can be used for rapid preliminary screening to determine whether global m⁶A levels are associated with specific biological processes, such as cell differentiation.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-deazaadenosine (3-DAA)	MCE	HY-W013332A	1 mg
Anti-m6A Monoclonal antibody	Proteintech	68055-1-Ig	50 μL
Bovine serum albumin (BSA, Fraction V)	BioFroxx	4240GR100	100 g
ChemiDoc MP Imaging System	BIO RAD	12003154
DMEM High Glucose	WISENT	319-005-CL	500 mL
DS-11 spectrophotometer	DeNOVIX	DS-11
EZ-press RNA Purification Kit	EZB	B0004DP	100 Preps
FBS- Superior quality	WISENT	086-150	500 mL
HRP-conjugated Goat Anti-Mouse IgG (H+L)	Beyotime	A0216	1 mL
Immobilon Western chemiluminescent HRP Substrate	Millipore	WBKLS0500	2 × 250 mL
Meclofenamic acid (MA)	MCE	HY-117275	1 mg
Methylene blue staining solution	Solarbio	G1300	0.1%, 100 mL
Nylon transfer membrane	LABSELECT	TM-NY-XS-45	7.4 × 8.5 cm, 0.45 μm, 10 PCS/BAG
PBS buffer (ready-to-use dry powder)	biosharp	BL601A	pH: 7.2-7.4, 2 L/ bag
Sodium dodecyl sulfate (SDS)	Sigma	L5750-500G	500 g
TBS buffer Premix powder	Sangon	A510025-0001	1EA (243.9 g powder)
TrypLE Express Enzyme	Gibco	12604-021	1×, 500 mL
Tween-20	Sangon	A600560-0500	500 mL
UV crosslinker	UVP	CL-1000