Name: Dot Blot Assay for Detecting Global N6-Methyladenosine RNA Modification Levels (Video) | JoVE Journal
Uploaded: 2026-02-06T15:00:00.000+00:00
Duration: 8 min 40 s
Description: Learn a dot blot assay for semi-quantitative assessment of global m6A RNA modification levels using cultured 143B cells. This rapid, cost-effective method enables routine screening and comparative analysis of epigenetic changes.

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5 minutes

This work was supported by grants from the National Nature Science Foundation (82174408, 82374477, and 82474535). Figure 1 was created with BioRender.com.

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
<ol>
	<li>Cell preparation
	<ol>
		<li>Take out 143B cells with stable SNHG21-knockdown (shSNHG21), negative control cells (shCtrl), and cells treated with 3-deazaadenosine (3-DAA; [100 &#181;M, 24 h]) and meclofenamic acid (MA; [100 &#181;M, 24 h]) from the 37 &#176;C incubator.</li>
		<li>Verify that the cell confluence is approximately 80-90% and the cells are in good condition under a microscope.</li>
		<li>Remove the culture medium, then rinse the cells twice with 1 mL of PBS.</li>
		<li>Remove PBS, then add 1 mL of trypsin per 6 cm dish.</li>
		<li>Gently shake to distribute the trypsin evenly and incubate for 1 min.</li>
		<li>Remove the trypsin, then add 1 mL of medium containing 10% FBS to terminate digestion.</li>
		<li>Pipette the cell suspension several times to disperse the cells.</li>
		<li>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.</li>
		<li>Centrifuge at 1000 &#215; g for 3 min, then remove the supernatant completely.</li>
	</ol>
	</li>
	<li>RNA extraction 
	NOTE: RNA extraction is performed using EZ-press RNA purification kit.
	<ol>
		<li>Add 600 &#181;L 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 &#215; 106 cells are used for RNA isolation.</li>
		<li>Add an equal volume of 100% ethanol and vortex thoroughly.</li>
		<li>Add 600 &#181;L 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.</li>
		<li>Centrifuge at 12,000 &#215; 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.</li>
		<li>Repeat the above steps 1.2.3-1.2.4 for the remaining mixture.</li>
		<li>Add 500 &#181;L of wash buffer (present in the RNA purification kit) to each spin column, let it stand at room temperature for 1 minute.</li>
		<li>Centrifuge at 12,000 &#215; g for 1 min and discard the liquid in the collection tube.</li>
		<li>Centrifuge the empty column again at 12,000 &#215; g for 1 min to remove residual liquid.</li>
		<li>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.</li>
		<li>Add 25-50 &#181;L of elution buffer (Enzyme-free water) to the center of the spin column.</li>
		<li>Incubate at room temperature for 2 min.</li>
		<li>Centrifuge at 12,000 &#215; g for 1 min to elute RNA.</li>
		<li>Discard the spin column and place the RNA on ice. 
		NOTE: Store RNA at -80 &#176;C until use.</li>
	</ol>
	</li>
	<li>RNA dilution
	<ol>
		<li>Measuring RNA concentration of 143B shSNHG21 cells,143B shCtrl cells, and cells treated with 3-DAA and MA using a DS-11 spectrophotometer.</li>
		<li>Prepare 1 &#181;g of total RNA from each cell line.</li>
		<li>Serially dilute RNA to 400 ng, 200 ng, and 100 ng with diethyl pyrocarbonate (DEPC)-treated water. 
		NOTE: Adjust concentration gradient as needed.</li>
	</ol>
	</li>
	<li>RNA denaturation
	<ol>
		<li>Heat diluted RNA samples at 95 &#176;C for 3 min.</li>
		<li>Immediately transfer the samples to ice to cool. 
		NOTE: Denatured RNA can be stored at 4 &#176;C for up to 1 week.</li>
	</ol>
	</li>
</ol>
2. Dot blot
<ol>
	<li>RNA spotting
	<ol>
		<li>Cut the positively charged nylon membrane to the desired size.</li>
		<li>Draw reference lines and label the membrane with the sample name.</li>
		<li>Mix the RNA thoroughly by gentle vortexing or pipetting.</li>
		<li>Spot 1-2 &#181;L 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 &#181;L per spot to prevent irregular diffusion.</li>
	</ol>
	</li>
	<li>RNA cross-Linking
	<ol>
		<li>Air-dry the membrane for 3 min.</li>
		<li>Perform RNA cross-linking by irradiating the membrane in a UV crosslinker at 254 nm, 120 mJ/cm&#178;. 
		NOTE: Incubate the membrane at 37 &#176;C for 30 min as an alternative option.</li>
		<li>Wash the membrane with 10 mL of 1x TBST on a rocking platform for 5 min to remove unbound RNA.</li>
	</ol>
	</li>
	<li>Membrane blocking
	<ol>
		<li>Prepare a 5% BSA solution. 
		NOTE: Prepare this solution fresh before use.</li>
		<li>Incubate the membrane in the blocking solution at room temperature for 2 h.</li>
	</ol>
	</li>
	<li>Antibody incubation
	<ol>
		<li>Incubate the membrane with anti-m&#8310;A antibody overnight at 4 &#176;C. 
		NOTE: Dilute antibody 1:1000 in 5% BSA solution before use. Incubate the membrane on a rocking platform with gentle shaking (50 rpm).</li>
		<li>Wash the membrane three times with 10 mL of 1x TBST on a rocking platform for 10 min.</li>
		<li>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.</li>
		<li>Wash the membrane three times with 10 mL of 1x TBST on a rocking platform for 10 min.</li>
	</ol>
	</li>
	<li>Chemiluminescence detection
	<ol>
		<li>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.</li>
		<li>Detect signal using a chemiluminescence imaging system.</li>
		<li>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%.</li>
	</ol>
	</li>
	<li>Methylene blue (MB) staining
	<ol>
		<li>Incubate the membrane in the MB staining solution for 1 min.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Quantification
<ol>
	<li>Grayscale value measurement
	<ol>
		<li>Import the image into the ImageJ software.</li>
		<li>Convert the image to 8-bit format using Image &#62; Type &#62; 8-bit.</li>
		<li>Subtract background with Process &#62; Subtract Background. 
		NOTE: Radius: 50.0 pixels, check Light background.</li>
		<li>Use the Rectangle tool to select the analysis area.</li>
		<li>Define lanes sequentially with Analyze &#62; Gels &#62; Select First Lane.</li>
		<li>Generate intensity profiles using Analyze &#62; Gels &#62; Plot Lanes and measure peaks.</li>
		<li>Measure the grayscale values of m&#8310;A and MB, respectively.</li>
	</ol>
	</li>
	<li>Copy the data into a table for analysis.</li>
	<li>Compare signal intensities to assess global m&#8310;A modification levels.</li>
</ol>

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The authors report no conflicts of interest.

In this study, knockdown of SNHG21 in osteosarcoma 143B cells resulted in reduced m&#8310;A 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 m&#8310;A levels are associated with specific biological processes, such as cell differentiation...

N6-methyladenosine (m&#8310;A) 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 export1,2,3. Dysregulation of m&#8310;A modification has been implicated in diverse physiological and pathological processes, including tumor initiation, progression, and metastasis4,5. In osteosarcoma, elevated m&#8310;A levels have been observed in both cell lines and tissues, suggesting a role for aberrant RNA methylation in disease biology6,7. Therefore, systematic assessment of global m&#8310;A 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 m&#8310;A modifications8,9. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)10 and enzyme-linked immunosorbent assay (ELISA)-based assays11 are both used to measure global m&#8310;A 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, m&#8310;A-seq14, and m&#8310;A-SAC-seq15 are sequencing-based approaches, which can precisely map m&#8310;A methylation sites; however, they are expensive and require extensive bioinformatics analysis.
The m&#8310;A dot blot assay offers a straightforward, rapid, and cost-effective method for determining global m&#8310;A modification levels in RNA. As a semi-quantitative immunoassay, its primary advantage lies in generating an overall view of m&#8310;A abundance without the need for specialized equipment or complex bioinformatic analysis. This makes the m&#8310;A 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.

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

dot blot assay for detecting global n6-methyladenosine rna modification levels

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

N6-methyladenosine (m&#8310;A) 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 m&#8310;A 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 m&#8310;A 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-m&#8310;A 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 m&#8310;A dot blot assay in both basic research and translational applications.

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<sup...

Here, we present a protocol to semi-quantitatively assess global m&#8310;A levels using dot blot. Total RNA is extracted, denatured, spotted on a nylon membrane, probed with anti-m&#8310;A antibody, and visualized by chemiluminescence. Signal intensity, quantified by ImageJ grayscale analysis, reflects relative methylation abundance, providing a reproducible workflow for research.

N6-methyladenosine (m&#8310;A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA) and serves as a key regulator of ...

Our protocol enables same quantitative assessment of global m6A levels using dot blood. This protocol can be applied to routine screening and the preliminary functional verification of m6A. To begin, remove cell culture dishes containing 143B cells with stable SNHG 21 knockdown negative control cells and cells treated with 3-deazaadenosine and meclofenamic acid from the incubator set to 37 degrees Celsius.Verify that cell confluence is approximately 80 to 90%under a microscope. Rinse the cells twice with 1 ml of PBS after removing the culture medium. Replace the PBS with 1 ml of trypsin per 6 cm dish.Gently shake the dish to distribute the trypsin evenly and incubate at room temperature for one minute. Next, replace the trypsin solution with 1 mi of medium containing 10%FBS to terminate digestion. Pipette the cell suspension several times to disperse the cells.Transfer the cell suspension to a 1.5 ml ribonuclease-free micro centrifuge tube. Centrifuge the tube at 1000 G for three minutes and pipette out the supernatant. For RNA extraction, add 600 microliters of lysis buffer from the RNA purification kit to the cell pellet.Vortex thoroughly to lyse the cells and allow the solution to stand for five minutes. After five minutes, add an equal volume of 100%ethanol to the lysate and vortex thoroughly. Now pipette 600 microliters of the mixture onto a spin column assembled with a collection tube.Centrifuge the column at 12, 000 G for one minute. Discard the liquid from the collection tube. After centrifuging the remaining mixture, add 500 microliters of wash buffer from the RNA purification kit to the spin column and incubate at room temperature for one minute.Then centrifuge the column at 12, 000 G for one minute. Discard the liquid from the collection tube and centrifuge the empty column again to remove residual liquid. Now transfer the spin column to a new 1.5 milliliter ribonuclease-free tube.Open the lid to air dry for two minutes. Add 25 to 50 microliters of enzyme free water to the center of the spin column. After a two-minute incubation at room temperature, centrifuge the column at 12, 000 G for one minute to elute RNA.Discard the spin column and place the eluted RNA on ice. Next, measure RNA concentration of control and knockdown samples using a spectrophotometer. Prepare one microgram of total RNA from each cell line.Then serially dilute RNA to 400 nanograms, 200 nanograms and 100 nanograms using diethyl pyrocarbonate-treated water. Heat the diluted RNA samples at 95 degrees Celsius for three minutes. Immediately transfer the tubes to ice to cool.To perform dot blotting, first draw reference lines on the positively charged nylon membrane. Label the membrane with sample names. Now, mix each RNA sample thoroughly by gentle vortexing or pipetting.Then spot 1 to 2 microliters of each RNA sample onto designated positions on the membrane. Irradiate the membrane in a ultraviolet crosslinker at 254 nanometers and 120 millijoules per square centimeter to perform RNA cross-linking. Then transfer the membrane into a dish containing 10 ml tris-buffered saline with Tween 20 and incubate it on a rocking platform for five minutes.Next, prepare a fresh 5%bovine serum albumin blocking solution just before use and incubate the membrane in the blocking solution at room temperature for two hours. Then incubate the membrane with anti m6A antibody at 4 degrees Celsius overnight on a rocking platform set to 50 revolutions per minute. The next day, wash the membrane three times with 10 ml of tris-buffered saline with Tween 20 for 10 minutes each on a rocking platform.Then incubate the membrane with horseradish peroxidase-conjugated secondary antibody for one hour at room temperature. After washing the membrane three times again, evenly apply chemiluminescent horseradish peroxidase substrate dropwise onto the membrane and incubate for one minute at room temperature with gentle rocking. Detect the signal using a Chemiluminescence Imaging System.Perform an initial pre-scan using the auto exposure function. For methylene blue staining, incubate the membrane in methylene blue staining solution for one minute. Wash the membrane three times with 10 ml of tris-buffered saline with Tween 20 for five minutes each.Image as a loading control. Destain the membrane using a solution containing 1%sodium dodecyl sulfate on a rocking platform for 15 minutes. Next, import the acquired image into the Image J software and adjust the format.Subtract the background by selecting Process and choosing Subtract Background. Set the radius to 50.0 pixels and enable light background. Use the rectangle tool to select the analysis area.Then define lanes sequentially by selecting Analyze, Gels and choosing Select First Lane. Then sequentially click on Analyze, Gels, Plot Lanes, and then measure peaks to generate intensity profiles. Measure the gray scale values of N6-methyladenosine and methylene blue signals.Copy the quantified data into a table for analysis. Compare signal intensities to assess global N6-methyladenosine modification levels. Treatment with the methyl transferase inhibitor 3-deazaadenosine markedly decreased the global N6-methyladenosine signal in 143B cells.Knockdown of SNHG 21 produced a comparable reduction in baseline global N6-methyladenosine levels. Additional treatment with 3-deazaadenosine further decreased the global N6-methyladenosine signal in SHSNHG 21 cells. Treatment with the demethylase inhibitor meclofenamic acid significantly increased global N6-methyladenosine levels in 143B cells.Despite lower basal N6-methyladenosine levels, SHSNHG 21 cells exhibited a robust increase in N6-methyladenosine, following meclofenamic acid treatment. The most important challenges include the membrane selection, precise RNA dilution and the uniform both size. Perform sequencing for precise state specific identification and apply mass spectrum trait to achieve comprehensive global quantitative analysis.Future studies can investigate m6A mechanism in diseases development and progression.

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Medicine

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