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 tissues, suggesting a role for aberrant RNA methylation in disease biology^6,7. Therefore, systematic assessment of global m⁶A abundance, together with its changes in osteosarcoma-associated transcripts and regulatory pathways, is essential for clarifying the biological mechanisms and clinical implications of RNA methylation in osteosarcoma.

Several analytical methods are available to study m⁶A modifications^8,9. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)¹⁰ and enzyme-linked immunosorbent assay (ELISA)-based assays¹¹ are both used to measure global m⁶A levels. The former offers high sensitivity but requires specialized equipment and expertise, while the latter gives relative m⁶A levels and contributes to a higher cost than the dot blot assay. MeRIP-qPCR enables targeted assessment of specific transcripts, but it is strongly influenced by immunoprecipitation efficiency¹². MeRIP-seq¹³, m⁶A-seq¹⁴, and m⁶A-SAC-seq¹⁵ are sequencing-based approaches, which can precisely map m⁶A methylation sites; however, they are expensive and require extensive bioinformatics analysis.

The m⁶A dot blot assay offers a straightforward, rapid, and cost-effective method for determining global m⁶A modification levels in RNA. As a semi-quantitative immunoassay, its primary advantage lies in generating an overall view of m⁶A abundance without the need for specialized equipment or complex bioinformatic analysis. This makes the m⁶A dot blot assay a valuable tool for routine screening, preliminary functional validation, and clinical research, as well as a practical complement to advanced sequencing and mass spectrometry-based approaches. The present protocol offers a detailed step-by-step workflow, ensuring reproducible detection and broad applicability.

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 incubator.
   2. Verify that the cell confluence is approximately 80-90% and the cells are in good condition under a microscope.
   3. Remove the culture medium, then rinse the cells twice with 1 mL of PBS.
   4. Remove PBS, then add 1 mL of trypsin per 6 cm dish.
   5. Gently shake to distribute the trypsin evenly and incubate for 1 min.
   6. Remove the trypsin, then add 1 mL of medium containing 10% FBS to terminate digestion.
   7. Pipette the cell suspension several times to disperse the cells.
   8. Transfer the cell suspension to a 1.5 mL RNase-free microcentrifuge tube.
      NOTE: Label the tube before use. Steps 1.1.3 through 1.1.8 must be performed under sterile conditions.
   9. Centrifuge at 1000 × g for 3 min, then remove the supernatant completely.
2. RNA extraction
   NOTE: RNA extraction is performed using EZ-press RNA purification kit.
   1. Add 600 µL of lysis buffer (present in the RNA purification kit) to the pellet and vortex thoroughly to completely lyse the cells, and let it stand for 5 min.
      NOTE: Approximately 2 × 10⁶ cells are used for RNA isolation.
   2. Add an equal volume of 100% ethanol and vortex thoroughly.
   3. Add 600 µL of the mixture onto a spin column assembled with a collection tube.
      NOTE: Assemble the spin column and collection tube before use, and label the tube before use.
   4. Centrifuge at 12,000 × g for 1 min and discard the liquid in the collection tube.
      NOTE: Avoid contact between the column bottom and the liquid or the operator.
   5. Repeat the above steps 1.2.3-1.2.4 for the remaining mixture.
   6. Add 500 µL of wash buffer (present in the RNA purification kit) to each spin column, let it stand at room temperature for 1 minute.
   7. Centrifuge at 12,000 × g for 1 min and discard the liquid in the collection tube.
   8. Centrifuge the empty column again at 12,000 × g for 1 min to remove residual liquid.
   9. Transfer the spin column to a new 1.5 mL RNase-free tube, open the lid, and air-dry for 2 min.
      NOTE: Label the tube before use.
   10. Add 25-50 µL of elution buffer (Enzyme-free water) to the center of the spin column.
   11. Incubate at room temperature for 2 min.
   12. Centrifuge at 12,000 × g for 1 min to elute RNA.
   13. Discard the spin column and place the RNA on ice.
       NOTE: Store RNA at -80 °C until use.
3. RNA dilution
   1. Measuring RNA concentration of 143B shSNHG21 cells,143B shCtrl cells, and cells treated with 3-DAA and MA using a DS-11 spectrophotometer.
   2. Prepare 1 µg of total RNA from each cell line.
   3. Serially dilute RNA to 400 ng, 200 ng, and 100 ng with diethyl pyrocarbonate (DEPC)-treated water.
      NOTE: Adjust concentration gradient as needed.
4. RNA denaturation
   1. Heat diluted RNA samples at 95 °C for 3 min.
   2. Immediately transfer the samples to ice to cool.
      NOTE: Denatured RNA can be stored at 4 °C for up to 1 week.

2. Dot blot

1. RNA spotting
   1. Cut the positively charged nylon membrane to the desired size.
   2. Draw reference lines and label the membrane with the sample name.
   3. Mix the RNA thoroughly by gentle vortexing or pipetting.
   4. Spot 1-2 µL of each RNA sample onto the designated positions on the membrane.
      NOTE: Use RNase-free pipette tips at all times. Avoid direct contact between the pipette tip and the membrane. Allow RNA to diffuse naturally on the membrane. Use a new pipette tip for each sample. Do not exceed 2 µL per spot to prevent irregular diffusion.
2. RNA cross-Linking
   1. Air-dry the membrane for 3 min.
   2. Perform RNA cross-linking by irradiating the membrane in a UV crosslinker at 254 nm, 120 mJ/cm².
      NOTE: Incubate the membrane at 37 °C for 30 min as an alternative option.
   3. Wash the membrane with 10 mL of 1x TBST on a rocking platform for 5 min to remove unbound RNA.
3. Membrane blocking
   1. Prepare a 5% BSA solution.
      NOTE: Prepare this solution fresh before use.
   2. Incubate the membrane in the blocking solution at room temperature for 2 h.
4. Antibody incubation
   1. Incubate the membrane with anti-m⁶A antibody overnight at 4 °C.
      NOTE: Dilute antibody 1:1000 in 5% BSA solution before use. Incubate the membrane on a rocking platform with gentle shaking (50 rpm).
   2. Wash the membrane three times with 10 mL of 1x TBST on a rocking platform for 10 min.
   3. Incubate the membrane with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature.
      NOTE: Dilute antibody 1:1000 in 5% BSA solution before use.
   4. Wash the membrane three times with 10 mL of 1x TBST on a rocking platform for 10 min.
5. Chemiluminescence detection
   1. Evenly apply the chemiluminescent HRP substrate dropwise onto the membrane.
      NOTE: Incubate the membrane with substrate for 1 min at room temperature with gentle rocking.
   2. Detect signal using a chemiluminescence imaging system.
   3. Use an auto-exposure function in the chemiluminescence imaging system as an initial pre-scan.
      NOTE: Accept auto-time only if the brightest signal is less than 65% saturated; otherwise, halve the exposure and reacquire until the peak signal falls within 30-60%.
6. Methylene blue (MB) staining
   1. Incubate the membrane in the MB staining solution for 1 min.
   2. Wash the membrane three times with 10 mL of 1x TBST on a rocking platform for 5 min, and image as a loading control.
      NOTE: Stain the same membrane used for chemiluminescence. Avoid disturbing sediment in the staining solution. Wear protective clothing and a mask, as MB is difficult to remove.
   3. Destain the MB-blotted membrane after imaging using a solution containing 1% SDS on a rocking platform for 15 min to facilitate documentation or potential re-probing.

3. Quantification

1. Grayscale value measurement
   1. Import the image into the ImageJ software.
   2. Convert the image to 8-bit format using Image > Type > 8-bit.
   3. Subtract background with Process > Subtract Background.
      NOTE: Radius: 50.0 pixels, check Light background.
   4. Use the Rectangle tool to select the analysis area.
   5. Define lanes sequentially with Analyze > Gels > Select First Lane.
   6. Generate intensity profiles using Analyze > Gels > Plot Lanes and measure peaks.
   7. Measure the grayscale values of m⁶A and MB, respectively.
2. Copy the data into a table for analysis.
3. Compare signal intensities to assess global m⁶A modification levels.

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