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 of the glass plates and electrophoresis unit
<ol>
	<li>Wash all the apparatus required for the gel electrophoresis and electro-blotting with detergent. Gently scrub them using a soft sponge to remove residual buffer and acrylamide, rinse with water and allow them to dry.</li>
	<li>Assemble both the glass plates together and place them firmly on the sponge. Use a 1 mm thick plate for this setup. Make sure that the plates are at the same level to avoid leakage.</li>
	<li>To the 10 mL gel mix, add 8 &#181;L of TEMED and 80 &#181;L of freshly prepared 10% (w/v) ammonium persulfate solution.</li>
	<li>Without delay, gently mix and pour this in between the assembled glass plates. Place the comb carefully. Avoid generating air-bubbles in this step.</li>
	<li>Allow the gel to polymerize for approximately 45 min.</li>
	<li>Wash the polymerized gel with sterile water before placing inside the gel-running setup.</li>
	<li>Assemble the plates inside the running cassette and remove the comb gently.</li>
	<li>Pour freshly prepared sterile 1x TBE, pH 8.2 into the tank.</li>
	<li>Wash the wells carefully by pipetting the buffer to remove salt crystals. This step helps the RNA sample to run uniformly across the gel.</li>
	<li>Perform a pre-run at 80 V for 30 min.</li>
</ol>
3. Preparation of loading dye and samples
<ol>
	<li>For 10 mL of gel loading dye, weigh 5 mg of bromophenol blue, 5 mg of xylene cyanol, add 10 mL of deionized formamide carefully and mix them well.</li>
	<li>Aliquot 10 &#181;g of total RNA into a sterile 1.5 mL tube and dry the samples using a speedvac. Do not over-dry the samples.</li>
	<li>Resuspend the RNA samples in 8 &#181;L of loading.</li>
	<li>Heat the samples at 98 &#176;C for 2 min, cool for 1 min at RT, vortex and spin the samples, for 3 times. This step is essential for proper resuspension and in turn this helps in equal loading of the samples.</li>
</ol>
4. Gel electrophoresis
<ol>
	<li>Stop the pre-run and wash the wells before sample loading.</li>
	<li>Heat the samples at 98 &#176;C for 1 min and load the samples hot into the well using capillary tips. Insert the tip to the bottom of the well, so that sample occupies one thin layer in the well.</li>
	<li>Complete loading of all the samples. Include to load RNA decade marker.</li>
	<li>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.</li>
</ol>
5. Preparation for electro-blotting
<ol>
	<li>Cut the N+nylon membrane to the dimensions of glass plate and label the membrane at its top-right corner with a HB pencil.</li>
	<li>Gently place the membrane on surface of sterile water, facing the labeled side towards water surface. Pre-soak the membrane for 15 min.</li>
	<li>Cut 4 pieces of blotting paper I to the dimensions of fiber pad.</li>
	<li>Prepare the gel sandwich for placing the gray side of the cassette down in a clean tray.</li>
	<li>Pre-wet the fiber pad and place it over the cassette. Remove air bubbles.</li>
	<li>Pre-wet one piece of blotting paper in 1x TBE and place 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.</li>
	<li>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.</li>
	<li>Gently dip the pre-soaked membrane in 1x TBE and place it over the gel, facing the labelled side down. Roll out to remove the air bubbles.</li>
	<li>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 sandwich setup and remove air bubbles.</li>
	<li>Complete the sandwich by placing a pre-wet fiber pad over the setup and firmly close the cassette.</li>
	<li>Place the transblot cassette in the module and fill 1x TBE, pH 8.2 up to the blotting mark.</li>
	<li>Transfer at 10 V, overnight at 4 &#176;C.</li>
	<li>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 &#181;joules/cm&#178;). The cross-linked blot maybe stored at 4 &#176;C for further hybridization.</li>
</ol>
6. Preparation of radiolabeled probe
<ol>
	<li>Design a probe that is completely complementary to the small RNA which is to be detected.</li>
	<li>End label the probe using &#435;P32ATP (obtained from BRIT) at its 5&#8217; end by combining the components as per recipe provided in Table 1.</li>
	<li>Incubate the above reaction mixture at 37 &#176;C for 30 min.</li>
	<li>Separate un-labelled &#435;P32ATP from probe by using a Sephadex G-25 column according to manufacturer&#8217;s protocol.</li>
</ol>
7. Hybridization of the blot
<ol>
	<li>Place the crossed-linked blot, RNA side facing top inside a hybridization bottle.</li>
	<li>Vigorously mix the ultra-sensitive hybridization buffer, add 10 mL of it and incubate the blot inside a hybridization oven maintained at 35 &#176;C, with rotation.</li>
	<li>Perform pre-hybridization for 20 min.</li>
	<li>Remove the bottle from the oven and add the labelled probe into the hybridization buffer gently.</li>
	<li>Hybridize the blot at 35 &#176;C, with rotation for 12 h.</li>
	<li>After hybridization carefully transfer the hybridization solution into 15 mL tube. This solution can be stored at 4 &#176;C until re-use.</li>
	<li>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.</li>
	<li>Incubate the blot with 2x SSC buffer plus 0.5% SDS for 35 &#176;C, with rotation for 30 min.</li>
	<li>Wash the blot again with 0.5x SSC buffer plus 0.5% SDS for 35&#176;C, with rotation for 30 min.</li>
	<li>Place the blot inside a hybridization cover, remove the excess buffer and seal it.</li>
	<li>Expose it to a radiation free-phosphor imager screen overnight inside a cassette.</li>
	<li>Detect the hybridization signal using biomolecular imager and analyze the results using suitable software.</li>
	<li>Strip the blot by incubating it at 80 &#176;C, with rotation for 30 min with 0.5 X SSC buffer plus 0.1% SDS and 0.1 X SSC buffer plus 0.1% SDS for 30 min.</li>
</ol>

<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>

No conflicts of interests declared.

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&amp;rsquo;,N&amp;rsquo;-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 ...

rna-blot-analysis-for-detection-quantification-plant

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

10.5K Views.  Tata Institute of Fundamental Research. This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

Video: RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

Genetics

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Immunology and Infection

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools.

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in ...

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method.

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

The methylated RNA immunoprecipitation assay is an antibody-based method used to enrich for methylated RNA fragments. Coupled with deep sequencing, it leads to the identification of transcripts carrying the m5C modification.

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Medicine

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Biology

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

mirMachine: A One-Stop Shop for Plant miRNA Annotation

Of different types of noncoding RNAs, microRNAs (miRNAs) have arguably been in the spotlight over the last decade. As post-transcriptional regulators of gene expression, miRNAs play key roles in various cellular pathways, including both development and response to a/biotic stress, such as drought and diseases. Having high-quality reference genome sequences enabled identification and annotation of miRNAs in several plant species, where miRNA sequences are highly conserved. As computational miRNA identification and annotation processes are mostly error-prone processes, homology-based predictions increase prediction accuracy. We developed and have improved the miRNA annotation pipeline, SUmir, in the last decade, which has been used for several plant genomes since then. 
This study presents a fully automated, new miRNA pipeline, mirMachine (miRNA Machine), by (i) adding an additional filtering step on the secondary structure predictions, (ii) making it fully automated, and (iii) introducing new options to predict either known miRNA based on homology or novel miRNAs based on small RNA sequencing reads using the previous pipeline. The new miRNA pipeline, mirMachine, was tested using The Arabidopsis Information Resource, TAIR10, release of the Arabidopsis genome and the International Wheat Genome Sequencing Consortium (IWGSC) wheat reference genome v2.

Herein, we present a new and fully automated miRNA pipeline, mirMachine that 1) can identify known and novel miRNAs more accurately and 2) is fully automated and freely available. Users can now execute a short submission script to run the fully automated mirMachine pipeline.

Of different types of noncoding RNAs, microRNAs (miRNAs) have arguably been in the spotlight over the last decade. As post-transcriptional regulators of ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

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Methylated RNA Immunoprecipitation Assay to Study m⁵C Modification in Arabidopsis

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

mirMachine: A One-Stop Shop for Plant miRNA Annotation