A Method for Measuring RNA N⁶-methyladenosine Modifications in Cells and Tissues

The overall goal of the modified northern blotting technique is to measure N6-methyladenosine, n6A modifications in RNA and detect modifications in diverse RNAs. This method can help answer key questions in the role of RNA epigenetics such as N6-methyladenosine modifications. The main advantage of this technique is that it provides the effective separation of RNA, better standardization for experimental designs and a clear delineation of n6A modification in RNAs.

This method can provide insight into n6A modifications in RNA and it can also be applied to other RNA modifications with different detection antibodies. To begin the experiment, spray ribonuclease decontamination solution onto paper towels and wipe down the pipettes and lab bench surfaces. Wet the paper towels with nuclease free water and wipe down the pipettes and lab bench surfaces again.

Obtain RNA isolation solution maintained at four degrees Celsius and add one milliliter of the solution to about 75 milligrams of mouse tissue. Use an overhead stirrer to homogenize the tissue. Next, add 100 microliters of 1-Bromo-3-chloropropane or BCP per one milliliter of sample homogenate.

Shake the tubes vigorously for 15 seconds and proceed to total RNA isolation. Using a spectrophotometer, determine the RNA concentration by noting the absorbance at 260 nanometers and 280 nanometers. Check the RNA sample quality by performing 0.8%agarose gel electrophoresis.

Rinse all of the electrophoresis equipment with diethylpyrocarbonate or DEPC water. Combine 2.5 grams of agarose, then 215 milliliters of DEPC water in an Erlenmeyer flask and completely melt the mixture in a microwave. After the solution has melted, add 12.5 milliliters of 10X MOPS buffer.

And 22.5 milliliters of 37%formaldehyde to the flask. Then pour the solution into the electrophoresis apparatus with a 1.5 millimeter thick comb. Using a clean comb, push bubbles to the edges of the gel.

Allow the gel to solidify at room temperature. Next, use previously prepared 1X MOPS running buffer to rinse the wells of the gel and pre-run the gel. Load the RNA samples into the wells of the gel, then run the gel.

The next day, examine and photograph the gel under a UV light. To prepare the transfer, cut the gel and remove the unused portion. Prepare 500 milliliters of 10X saline sodium citrate or SSC buffer.

Wash the gel twice with 10X SSC buffer. Cut one filter paper sheet large enough to cover the gel. Cut four pieces of filter paper to the same size as the gel.

Next, cut one sheet of positively charged nylon membrane to the same size as the gel. Mark a notch on the gel, and the membrane, as a marker to ensure the proper orientation. Soak the membrane in 2X SSC buffer.

Pour 500 milliliters of 10X SSC buffer into the transfer tray. Lay the large filter paper sheet across the glass plate and wet the filter paper with transfer buffer. Then use a pipette to roll out any bubbles.

Soak two pieces of pre-cut filter paper in transfer buffer and place them on the center of the filter paper. Remove any bubbles. Then lay the gel top side down on the filter paper and lay the membrane on top of the gel, being sure to align the notches of the gel and the membrane.

Next, place two pieces of pre-cut filter paper on top of the membrane. Remove any bubbles between the layers. Apply plastic wrap around the gel to ensure that the towels only soak up buffer passing through the gel and the membrane.

Then stack paper towels on top of the filter paper. Place an oversized glass plate on top of the towels and place a weight on top of the stack and allow the RNA to transfer from the gel to the membrane overnight. Following the transfer, keep the membrane damp in 2X SSC buffer.

Immerse two sheets of filter paper with 10X SSC buffer, then place the sheets into the UV cross linker. Make sure the RNA absorbed side is facing up, then place the membrane on top of the filter paper. Select auto cross link mode, and press the start button to initiate the irradiation.

Examine the membrane and the gel with a UV gel image catcher to confirm the RNA transfer from the gel to the membrane. Block the membrane in 5%non-fat milk in TBST buffer at room temperature for one hour. Incubate the membrane overnight in m6A antibody solution at four degrees Celsius.

The next day, wash the membrane three times with TBST buffer for 15 minutes. Next, incubate the membrane in the HRP conjugated donkey anti-rabbit antibody solution for one hour at room temperature. After one hour, wash the membrane three times with TBST buffer for 15 minutes.

Apply the enhanced chemiluminescence substrate. Capture the chemiluminescence with a digital imager. Finally, move onto m6A quantification and measure the relative m6A chemiluminescent intensity using image J.Mouse liver RNAs were sampled at four hour intervals and studied with the modified northern blotting technique.

The methylation of rRNAs, mRNAs, and small RNAs was clearly detected. Representative m6A blots were performed for total RNA and without rRNA from the livers of wild type mice. Once mastered, this technique can be done in four days if it is performed properly.

After its development, this technique paved the way for researchers in the field of RNA epigenetics to explore impacts on human physiology related to circadian rhythm and obesity.