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

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.