RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

Summary

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

Abstract

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

Introduction

The recent discovery of small regulatory RNAs, microRNAs, has led research in understanding them and their role in plants and animals¹. Long precursors of miRNAs are processed into 21 to 24 nt mature miRNAs by HYL1 and specific dicer-like proteins^2,3. A 22 nt miRNA can initiate cascade silencing by generating secondary siRNAs⁴. Studies have shown the role of miRNAs and secondary siRNAs in development, cell fate and stress responses^5,6.

Northern hybridization is an experimental ....

Protocol

1. Preparation of a 15% denaturing polyacrylamide gel

1. Weigh and add 4.8 g of urea, add 3.75 mL of 40% acrylamide: bisacrylamide (19:1) solution and 1 mL of 10x TBE pH 8.2 into a sterile 50 mL tube.
2. Dissolve the urea using a water bath set at 60 °C into a clear solution.
3. Make-up the volume to 10 mL using freshly autoclaved sterile water and cool the gel mix to room temperature.
4. Prepare fresh 10% (w/v) ammonium persulfate solution.

2. Assembly of the glass plates and electrophoresis unit

1. Wash all the apparatus required for the gel electrophoresis and electro-blotting with deter....

Results

In this demonstration, we have detected and quantified the expression of miR397 in different tissues of indica rice var whiteponni (Figure 1). miR397 is a 22 nt miRNA and conserved miRNA. The expression of miR397 can be detected in all the tested samples. As per the next-generation sequencing data, sample 1 (seedling tissue) has miR397 at 5 reads per million (rpm). We detected its signal comfortably, indicating that the method is very sensitive and can be used to detect eve.......

Discussion

This method can be extensively used for detection and quantification of small RNAs including less abundant miRNAs. The protocol mainly describes the steps for denaturing the total RNA in a loading buffer, size separation by gel electrophoresis, transfer of RNA to a membrane, cross-link the RNA onto membrane and hybridize using desired radiolabeled oligo probes.

The critical step for any blotting experiment is the use of good quality RNA for sample preparation. Before loading the gels, one must.......

Materials

List of materials used in this article

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