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

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.

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.

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 m6 A methylation (Figure 2). As shown in Figure 2A, treatment with the methyltransferase inhibitor 3-DAA (100 µM, 24 h) markedly decreased the global m6 A signal. Knockdown of SNHG21 alone produced a comparable reduction in baseline m6 A levels, and additional 3-DAA treatment further decreased the signal in shSNHG21 cells. These results demonstrate that the assay reliably detects progressive decreases in global m6 A abundance (Figure 2A,C,D).

We next examined whether the assay could sensitively capture increases in m6 A levels by blocking the demethylation pathway (Figure 3). As shown in Figure 3A, treatment with the demethylase inhibitor MA (100 µM, 24 h) significantly elevated global m6 A levels. Notably, despite their lower basal m6 A signal, shSNHG21 cells exhibited a robust increase in m6 A upon MA treatment, confirming the capacity of this assay to detect upward changes across a broad dynamic range (Figure 3A,C,D).

RNA quality correlates linearly with chemiluminescence signal
Grayscale intensities of the chemiluminescent signals were quantified using ImageJ and analyzed with GraphPad software. The dot blot signal increased proportionally with RNA input, yielding linear regression coefficients (R²) ranging from 0.93 to 0.99 across the same group (Supplementary File 1). One-way ANOVA revealed statistically significant differences among the tested RNA concentration groups in the same group (P < 0.05; Figure 2C, Figure 3C, and Supplementary File 1). At an RNA input of 400 ng, differences between experimental groups remained statistically significant (P < 0.05; Figure 2D and Figure 3D).

Grayscale intensities of the chemiluminescent signals were quantified using ImageJ and analyzed with GraphPad software. MB staining confirmed equal RNA loading within each concentration gradient (P > 0.05; Figure 2B, Figure 3B, and Supplementary File 1). However, because of the dark background in MB staining, which interferes with quantitative normalization, normalization is no longer performed.

Specificity control
To assess signal specificity, a technical control was included in which membranes were incubated with the HRP-conjugated secondary antibody alone. No detectable chemiluminescent signal was observed under these conditions (Figure 4), indicating that the detected signal is strictly dependent on the primary anti-m6 A antibody and not attributable to non-specific binding or artifacts in the detection system.

Collectively, these results demonstrate that the dot blot assay specifically, linearly, and sensitively detects both increases and decreases in global m6 A levels, supporting its utility as a rapid, semi-quantitative screening tool for studies in RNA epigenetics.

Figure 1: m6 A dot blot assay flow chart. Created with BioRender.com. Please click here to view a larger version of this figure.

Figure 2: Dot blot analysis of global m6 A levels following SNHG21 knockdown and 3-DAA treatment. (A) 143B shCtrl and shSNHG21 cells were treated with vehicle (0.1% DMSO) or the m6 A methyltransferase inhibitor (3-DAA; 100 µM) for 24 h. Total RNA was isolated, serially diluted (400, 200, and 100 ng), spotted onto a nylon membrane, and probed with an anti-m6 A antibody.(B) MB staining of the membrane confirms equal RNA loading across each concentration gradient.(C) Quantification of chemiluminescent signals demonstrates a linear relationship between m6 A signal intensity and RNA input (mean ±± SD, n = 3).(D) Quantitative comparison of m6 A signal intensity at 400 ng RNA input (mean ±± SD, n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey's multiple-comparison test (*P < 0.05, **P < 0.01, ***P < 0.001). Please click here to view a larger version of this figure.

Figure 3: Dot blot analysis of global m6 A levels following SNHG21 knockdown and MA treatment. (A) 143B shCtrl and shSNHG21 cells were treated with vehicle (0.1% DMSO) or the m6 A demethylase inhibitor (MA; 100 µM) for 24 h. Total RNA was isolated, serially diluted (400, 200, and 100 ng), spotted onto a nylon membrane, and probed with an anti-m6 A antibody. (B) MB staining of the membrane confirms equal RNA loading across each concentration gradient. (C) Quantification of chemiluminescent signals demonstrates a linear relationship between m6 A signal intensity and RNA input (mean ±± SD, n = 3). (D) Quantitative comparison of m6 A signal intensity at 400 ng RNA input (mean ±± SD, n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey's multiple-comparison test (*P < 0.05, **P < 0.01, ***P < 0.001). Please click here to view a larger version of this figure.

Figure 4: Secondary antibody-only control for dot blot specificity. (A) 143B shCtrl and shSNHG21 cells were treated with vehicle (0.1% DMSO) or the m6 A methyltransferase inhibitor (3-DAA; 100 µM) for 24 h. Total RNA was isolated, serially diluted (400, 200, and 100 ng), and spotted onto a nylon membrane. Membranes were incubated with the HRP-conjugated secondary antibody alone, without the primary anti-m6 A antibody. No detectable chemiluminescent signal was observed, confirming the absence of non-specific binding.(B) MB staining of the membrane confirms equal RNA loading across each concentration gradient. Please click here to view a larger version of this figure.

Supplementary Table 1: Reagent formulations. Please click here to download this File.

Supplementary File 1: Statistical analysis and results for Figure 2B,C, and Figure 3B,C. Please click here to download this File.

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, stress response, or tumor progression¹⁶. It also serves to evaluate the functional roles of m⁶A regulators ("Writers," "Erasers," and "Readers") by assessing whether their manipulation leads to measurable changes in overall methylation abundance^17,18. 2) Analysis of specific RNA populations: This assay enables detection of m⁶A enrichment within defined RNA subsets, including mRNA, lncRNA, or rRNA, thereby providing insights into RNA class-specific methylation patterns¹⁹. 3) Clinical and translational applications: It offers a practical approach for comparing global m⁶A levels in clinical samples, such as tumor versus adjacent normal tissues, supporting biomarker development and disease association studies²⁰. 4) Complement to high-resolution techniques: Serves as a preparatory or supportive experiment for advanced methods such as MeRIP-seq, LC-MS/MS, providing a rapid and economical overview before committing to resource-intensive analyses⁸. Overall, the m⁶A dot blot assay provides a versatile, efficient, and economical platform for global m⁶A assessment, making it a valuable tool for preliminary screening, functional validation, clinical research, and as a complement to advanced sequencing- or mass spectrometry-based approaches.

Several technical considerations are crucial for reproducible and accurate results. First, a uniform spot size requires accurate RNA dilution, calibrated pipettes, and consistent sample loading speed. Second, Nylon membranes are recommended for their strong nucleic acid binding capacity, low background, and mechanical stability, providing excellent RNA retention²¹. Third, MB staining provides a simple and effective loading control, reflecting total RNA input and enabling reliable normalization across samples²². Attention to these steps ensures data quality and interpretability. In addition, the dilution series of RNA input should demonstrate a linear relationship with the signal intensity detected on the membrane. This linearity validates the accuracy of the experimental procedure and meets a fundamental requirement for robust semi-quantitative analysis.

While strict adherence to the key technical considerations outlined above is essential to ensure assay accuracy, several experimental parameters remain flexible and may be optimized according to specific experimental requirements. For instance, although an EZB kit was employed for RNA extraction in the present protocol, the dot blot assay itself is not restricted to a particular extraction method; any approach that yields high-quality, intact RNA is suitable²³. The MB staining step may be performed either as described in this protocol or repositioned to occur after the membrane cross-linking step (i.e., following step 2.2.3). Moreover, to minimize background signals and enhance the signal-to-noise ratio, several key detection parameters can be further optimized as needed. These include the RNA loading amount and dilution range, the membrane cross-linking strategy (e.g., UV irradiation or baking, together with the corresponding temperature and duration), the optimal working concentration of the antibody, the incubation times for blocking, primary and secondary antibodies, and MB staining, as well as the number and duration of membrane washing steps.

The dot blot assay has several inherent limitations. It is semi-quantitative, lacks single-nucleotide resolution, and does not provide information on modification patterns within individual transcripts²⁴. Its sensitivity is also influenced by RNA quality, loading consistency, and antibody specificity, restricting its application to global rather than site-specific analyses^25,26.

In conclusion, although the m⁶A dot blot assay cannot substitute for high-resolution sequencing or mass spectrometry, its simplicity, speed, and low cost make it well-suited for rapid assessment of m⁶A abundance. It serves as a practical and accessible method for preliminary screening and comparative studies, and it complements analytical techniques in RNA epigenetics research.