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

1. Gel electrophoresis

1. Stop the pre-run and wash the wells before sample loading.
2. Heat the samples at 98 °C for 1 min and load the samples hot into the well using capillary tips. Insert the tip into the bottom of the well so that the sample occupies one thin layer in the well.
3. Complete loading of all the samples. Include loading the RNA decade marker.
4. Run the gel at 80 V until the bromophenol blue dye runs almost completely. Bromophenol blue runs at 10 bp in a 15% denaturing acrylamide gel.

2. Preparation for electro-blotting

1. Cut the N+nylon membrane to the dimensions of a glass plate and label the membrane at its top-right corner with an HB pencil.
2. Gently place the membrane on the surface of sterile water, facing the labeled side towards the water surface. Pre-soak the membrane for 15 min.
3. Cut 4 pieces of blotting paper I into the dimensions of a fiber pad.
4. Prepare the gel sandwich by placing the gray side of the cassette down in a clean tray.
5. Pre-wet the fiber pad and place it over the cassette. Remove air bubbles.
6. Pre-wet one 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. Lay another piece of pre-wet blotting paper and remove air bubbles.
7. Carefully remove the gel from the running cassette and place it over the sandwich setup, such that the first loaded RNA sample is towards the right.
8. Gently dip the pre-soaked membrane in 1x TBE and place it over the gel, facing the labeled side down. Roll out to remove the air bubbles.
9. Dip a piece of blotting paper, lay it over the membrane, and remove the air bubbles. Dip another piece of blotting paper, place it over the sandwich setup, and remove air bubbles.
10. Complete the sandwich by placing a pre-wet fiber pad over the setup and firmly closing the cassette.
11. Place the transblot cassette in the module and fill 1x TBE, pH 8.2, up to the blotting mark.
12. Transfer at 10 V, overnight at 4 °C.
13. After transfer, place the damp membrane on a paper sheet and immediately cross-link the RNA to the membrane by irradiation with 254 nm UV light (120,000 µjoules/cm²). The cross-linked blot may be stored at 4 °C for further hybridization.

3. Preparation of radiolabeled probe

1. Design a probe that is completely complementary to the small RNA which is to be detected.
2. End label the probe using ƳP32ATP (obtained from BRIT) at its 5' end by combining the components as per the recipe provided in Table 1.
3. Incubate the above reaction mixture at 37 °C for 30 min.
4. Separate un-labeled ƳP32ATP from the probe by using a Sephadex G-25 column according to the manufacturer's protocol.

4.Hybridization of the blot

1. Place the crossed-linked blot, RNA side facing the top, inside a hybridization bottle.
2. Vigorously mix the ultra-sensitive hybridization buffer, add 10 mL of it, and incubate the blot inside a hybridization oven maintained at 35 °C, with rotation.
3. Perform pre-hybridization for 20 min.
4. Remove the bottle from the oven and gently add the labeled probe into the hybridization buffer.
5. Hybridize the blot at 35 °C, with rotation for 12 h.
6. After hybridization, carefully transfer the hybridization solution into 15 mL tube. This solution can be stored at 4 °C until re-use.
7. Perform a quick wash of the blot for 2 min to remove excess hybridization solution by adding 2x SSC buffer plus 0.5% SDS. Discard the solution.
8. Incubate the blot with 2x SSC buffer plus 0.5% SDS for 35 °C, with rotation for 30 min.
9. Wash the blot again with 0.5x SSC buffer plus 0.5% SDS for 35°C, with rotation for 30 min.
10. Place the blot inside a hybridization cover, remove the excess buffer, and seal it.
11. Expose it to a radiation-free-phosphor imager screen overnight inside a cassette.
12. Detect the hybridization signal using a biomolecular imager and analyze the results using suitable software.

Table 1: Table of Recipes