Northern Blot to Detect and Quantify Specific MicroRNA in Total RNA Extract of Plant Tissue

Stop the pre-run of the gel.

Wash the wells thoroughly before loading the sample to remove deposits of the urea inside the wells. Heat the resuspended RNA samples at 98 degrees for 1 minute. Load the samples hot into the wells using capillary tips. Avoid introducing air bubbles.

Complete loading of all the samples and assemble the lid. Run the gel at 80 volts for 3 hours, or until the bromophenol blue runs completely. Use positively-charged nylon membrane for the transfer. Cut it to the dimensions of the glass plate. Label the membrane at its top right corner. Gently place the membrane on the surface of sterile Milli-Q water. Make sure to place the label side downwards facing the water surface.

Take a clean tray and prepare gel sandwich for electrotransfer. Place the gray side of the cassette down. Pour 1X TBE slightly above the level of the cassette. Pre-wet the fiber pad in 1X TBE, and squeeze it to remove air bubbles. Cut two pieces of blotting paper to the size of fiber pad.

Pre-wet a piece of blotting paper in 1X TBE, and place it over the fiber pad. Remove air bubbles by rolling a plastic pipette over the paper. Pre-wet another piece of blotting paper in one 1X TBE and lay it over the cassette. Rollover to remove air bubbles. Now the sandwich setup is ready for electrotransfer.

After completion of electrophoresis, stop the run and remove the lid from the apparatus. Remove the running cassette from the setup. Take out the glass plates from the running cassette.

Carefully remove the gel from the assembly. Lay it over the blotting paper such that the first loaded RNA sample is towards your right. Gently dip the pre-soaked membrane in 1X TBE and place it over the gel facing the label side down. Do not allow the membrane and gel to dry. Roll over gently to remove air bubbles.

Dip one piece of blotting paper in 1X TBE, and lay it over the membrane. Remove air bubbles. Place another piece of blotting paper, and remove the air bubbles. Complete the sandwich by laying the fiber pad over the assembly. Close the cassette firmly.

Place the transblot cassette in the module. Fill the tank with 1X TBE, pH 8.2, up to the blotting mark. Close the lid of the apparatus and transfer at 10 volts overnight at 4 degrees or in a cold room. Keep the UV cross-linker ready and set it to 1,200 just before completion of electro-transfer.

After completion of transfer, remove the cassette from module. Place the damp membrane on a A4 sheet placing the marked side upwards. Crosslink the RNA to the membrane by irradiation with 254-nanometer UV light at 120 millijoules. The cross-linked blot may be stored at 4 degree or used for hybridization.

Design a probe that is completely complementary to the small RNA that has to be detected, and label the probe at its 5 prime end using PNK enzyme, and combine the components as shown. Incubate the reaction mixture at 37 degrees for 30 minutes.

After 30 minutes of incubation, use G-25 columns to separate unlabeled probes from the reaction mixture. For improved labeling of the probe, prepare the G-25 column before use. Place the blot, RNA side facing top, inside a hybridization bottle. Vigorously mix hybridization buffer before use. Add 10 ml of hybridization buffer, and place the hybridization bottle inside a hybridization oven maintained at 35 degrees with rotation.

Perform pre-hybridization for 20 to 30 minutes. After pre-hybridization, remove the bottle from oven. Add the labeled probe into the hybridization buffer gently. Make sure air bubbles are not created. Incubate the blot inside the oven at 35 degrees with rotation for 12 hours.

After hybridization, remove the hybridization buffer from the bottle. Perform a quick wash of the blots using wash buffer I. This step is performed to remove excess hybridization solution from the blots. Further, incubate the blots at 35 degrees for 30 minutes using wash buffer I. Perform another wash using buffer II at 35 degrees for 30 minutes.

After washing off the blot, place it inside a hybridization cover, remove excess buffer, and seal it. Place it inside a cassette, and expose it to a radiation-free phosphor imager screen for 12 hours. Detect the hybridization signal using Typhoon Biomolecular Imager and analyze the results using ImageJ software.