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

Abstract

Source: Tirumalai, V., et al. RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs. J. Vis. Exp. (2020)

In this video, we demonstrate the northern blot, a hybridization technique to detect and quantify microRNAs (miRNAs) from total RNA extracted from 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 gl....

Materials

List of materials used in this article

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