Name: rna blot analysis for the detection and quantification of plant micrornas
Uploaded: 2020-07-11T14:00:00
Description: Watch this Scientific Journal Video about RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs at JoVE.com

The authors acknowledge the access to radiation lab provided by the host institute and BRIT for radioisotope.&#160;PVS laboratory is supported by National Center for Biological Sciences, Tata Institute for Fundamental Research and grants (BT/PR12394/AGIII/103/891/2014; BT/IN/Swiss/47/JGK/2018-19; BT/PR25767/GET/119/151/2017) from Department of Biotechnology, Government of India. MP acknowledges DBT-Research Associateship, DBT, Government of India.

1. Preparation of a 15% denaturing polyacrylamide gel
<ol>
	<li>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.</li>
	<li>Dissolve the urea using a water bath set at 60 &#176;C into a clear solution.</li>
	<li>Make-up the volume to 10 mL using freshly autoclaved sterile water and cool the gel mix to room temperature.</li>
	<li>Prepare fresh 10% (w/v) ammonium persulfate solution.</li>
</ol>
2. Assembly ...

<ol>
	<li>Baulcombe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+in+plants.">RNA silencing in plants.</a> Nature. 431, 356-363 (2004).</li><li>Anushree, N., Shivaprasad, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Plant+miRNA+Biogenesis.">Regulation of Plant miRNA Biogenesis.</a> Proceedings of the Indian National Science Academy. 95, (2017).</li><li>Narjala, A., Nair, A., Tirumalai, V., Hari Sundar, G. V., Shivaprasad, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+sequence+signature+is+essential+for+robust+plant+miRNA+biogenesis.">A conserved sequence signature is essential for robust plant miRNA biogenesis.</a> Nucleic Acids Research. , (2020).</li><li>Chen, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=22-Nucleotide+RNAs+trigger+secondary+siRNA+biogenesis+in+plants.">22-Nucleotide RNAs trigger secondary siRNA biogenesis in plants.</a> Proceedings of the National Academy of Sciences of the United States of America. 107, 15269-15274 (2010).</li><li>Shivaprasad, P. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microRNA+superfamily+regulates+nucleotide+binding+site-leucine-rich+repeats+and+other+mRNAs.">A microRNA superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs.</a> Plant Cell. 24, 859-874 (2012).</li><li>Shivaprasad, P. V., Dunn, R. M., Santos, B. A., Bassett, A., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraordinary+transgressive+phenotypes+of+hybrid+tomato+are+influenced+by+epigenetics+and+small+silencing+RNAs.">Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs.</a> The EMBO Journal. 31, 257-266 (2012).</li><li>Akbergenov, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+geminivirus-derived+small+RNAs+in+different+plant+species.">Molecular characterization of geminivirus-derived small RNAs in different plant species.</a> Nucleic Acids Research. 34, 462-471 (2006).</li><li>Tirumalai, V., Swetha, C., Nair, A., Pandit, A., Shivaprasad, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR828+and+miR858+regulate+VvMYB114+to+promote+anthocyanin+and+flavonol+accumulation+in+grapes.">miR828 and miR858 regulate VvMYB114 to promote anthocyanin and flavonol accumulation in grapes.</a> Journal of Experimental Botany. , (2019).</li><li>Li, C., Zamore, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Small+RNAs+by+Northern+Hybridization.">Analysis of Small RNAs by Northern Hybridization.</a> Cold Spring Harbor Protocols. (8), (2018).</li><li>Swetha, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+domestication-related+phenotypes+in+indica+rice+are+due+to+loss+of+miRNA-mediated+laccase+silencing.">Major domestication-related phenotypes in indica rice are due to loss of miRNA-mediated laccase silencing.</a> The Plant Cell. , (2018).</li></ol>

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...

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 proteins2,3. 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 responses5,6.
Northern hybridization is an experimental method routinely employed to detect specific RNA molecules. This method customizes its use in the detection of approximately 19 -24 nt long small RNAs from a pool of total RNAs7. In this demonstration, we illustrate the use of this technique for the detection and quantification of miRNAs. This method uses labelling of probes using radioisotopes; thus, miRNA levels in the sample can be detected with increased sensitivity. Unlike PCR-based methods, this method ensures quantification of expression as well as size determination of the miRNAs. In this protocol, we show crucial steps that improve miRNA detection. We have modified steps in blotting and hybridization for obtaining high resolution signal detection of miRNAs. This technique can also be used for the detection other endogenous small RNAs such as siRNAs, tasi-secondary RNAs and snoRNAs.

<table>
	<tbody>
		<tr>
			<td>40% Acrylamide-bisacrylamide solution</td>
			<td>Sigma</td>
			<td>A9926</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulphate (APS)</td>
			<td>BioRad</td>
			<td>1610700</td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting paper</td>
			<td>whatmann blotting paper I</td>
			<td>1001-125</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma</td>
			<td>B5525-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary loading tips</td>
			<td>BioRad</td>
			<td>2239915</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized formamide</td>
			<td>Ambion</td>
			<td>AM9342</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating block</td>
			<td>Eppendorff</td>
			<td>5350</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybond N+nylon membrane</td>
			<td>GE</td>
			<td>RPN203B</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization bottle</td>
			<td>Sigma</td>
			<td>Z374792-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Oven</td>
			<td>Thermo Scientific</td>
			<td>1211V79</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#8217;,N&#8217;-Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma</td>
			<td>T7024-25ml</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosphorImager</td>
			<td>GE- Typhoon scanner</td>
			<td>29187194</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosphorImager screen and cassette</td>
			<td>GE healthcare</td>
			<td>GE28-9564-75</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Gilson, models: P20 and P10</td>
			<td>FA10002M, FA10003M</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic pipette</td>
			<td>Falcon</td>
			<td>357550</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyacrylamide gel apparatus</td>
			<td>BioRad</td>
			<td>1658003EDU</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex G-25 column</td>
			<td>GE healthcare</td>
			<td>27532501</td>
			<td></td>
		</tr>
		<tr>
			<td>Speed Vac Concentrator</td>
			<td>Thermo Scientific</td>
			<td>20-548-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinwin</td>
			<td>Tarsons</td>
			<td>1010</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase (PNK)</td>
			<td>NEB</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>Transblot apparatus</td>
			<td>BioRad</td>
			<td>1703946</td>
			<td></td>
		</tr>
		<tr>
			<td>ULTRAHyb hybridization buffer</td>
			<td>Ambion</td>
			<td>AM8670</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Fischer Scientific</td>
			<td>15985</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-crosslinker</td>
			<td>UVP</td>
			<td>CL-1000L</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Tarsons</td>
			<td>3020</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>NEOLAB</td>
			<td>D-8810</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene cyanol</td>
			<td>Sigma</td>
			<td>X4126-10G</td>
			<td></td>
		</tr>
	</tbody>
</table>

rna blot analysis for the detection and quantification of plant micrornas

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.

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...

Watch this Scientific Journal Video about RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs at JoVE.com

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

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

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 ...

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