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

Protocol

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

Buffer and solution	Recipe	Comments
10 X TBE	0.89M Tris buffer	pH should be set to 8.2 using acetic acid
	0.89M Boric acid
	30mM EDTA
Gel mix	15% acrylamide-bisacrylamide sol, 19:1	Gel mix should not contain urea crystals
	8M urea
	1X TBE
Gel-loading dye	0.05 %(w/v) bromophenol blue	Care should be taken while handling deionized formamide
	0.05 %(w/v) Xylene xyanol
	100 % deionized- formamide
Labeling of probe	PNK buffer (10X, 2 µL)	This mixture contains radioactive molecules, this step must be performed by trained personnel inside a radioactive lab
	PNK enzyme (1 µL)
	Oligo (100 µM, 0.1 µL)
	ƳP32 ATP (4 µL)
Wash Buffer - I	2X SSC
	0.5% (w/v) SDS
Wash Buffer - II	0.5X SSC
	0.5% (w/v) SDS
Stripping Buffer - I	0.5X SSC
	0.1% (w/v) SDS
Stripping Buffer - II	0.1X SSC
	0.1% (w/v) SDS

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Materials

Name	Company	Catalog Number	Comments
40% Acrylamide-bisacrylamide solution	Sigma	A9926	Chemical
Ammonium persulphate (APS)	BioRad	1610700	Chemical
Blotting paper	Whatman blotting paper I	1001-125	Assay
Bromophenol blue	Sigma	B5525-5G	Chemical
Capillary loading tips	BioRad	2239915	Plastic ware
Deionized formamide	Ambion	AM9342	Chemical
Heating block	Eppendorf	5350	Device
Hybond N+nylon membrane	GE	RPN203B	Assay
Hybridization bottle	Sigma	Z374792-1EA	Plastic ware
Hybridization Oven	Thermo Scientific	1211V79	Device
N,N,N',N'- Tetramethylethylenediamine (TEMED)	Sigma	T7024-25ml	Chemical
PhosphorImager	GE- Typhoon scanner	29187194	Device
PhosphorImager screen and cassette	GE healthcare	GE28-9564-75	Device
Pipettes	Gilson, models: P20 and P10	FA10002M, FA10003M	Plastic ware
Plastic pipette	Falcon	357550	Plastic ware
Polyacrylamide gel apparatus	BioRad	1658003EDU	Device
Sephadex G-25 column	GE healthcare	27532501	Device
Speed Vac Concentrator	Thermo Scientific	20-548-130	Device
Spinwin	Tarsons	1010	Device
T4 Polynucleotide Kinase (PNK)	NEB	M0201S	Assay
Transblot apparatus	BioRad	1703946	Device
ULTRAHyb hybridization buffer	Ambion	AM8670	Assay
Urea	Fischer Scientific	15985	Chemical
UV-crosslinker	UVP	CL-1000L	Device
Vortex	Tarsons	3020	Device
Water bath	NEOLAB	D-8810	Device
Xylene cyanol	Sigma	X4126-10G	Assay