We would like to thank Athanasios Papangelis, for critical comments on the manuscript. FM is supported by the Biotechnology and Biological Sciences Research Council (BBSRC; BB/M009424/1). IP is supported by the American Heart Association Scientist Development Grant (17SDG33060002).

Mouse heart tissue samples for this study were obtained in compliance with the relevant laws and institutional guidelines and were approved by Yale University Institutional Animal Care and Use Committee.
1. Solution Preparation
<ol>
	<li>Prepare RNase free ddH2O, by treating 5 L of ddH2O with 5 mL of 0.1% diethylpyrocarbonate (DEPC) overnight (O/N), at room temperature (RT). Autoclave (121&#160;&#176;C) to deactivate the DEPC. Use DEPC-treated ddH2O for the p...

<ol>
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miRNA transcript detection can be achieved through different techniques that vary in sensitivity, specificity and quantitative power. Here, we demonstrate the coupling of miRNA in situ hybridization with immunostaining and describe a protocol that allows for concurrent assessment of the expression levels of miRNA and protein molecules, on the same heart sections. We first show how to perform in situ hybridization of DIG-labeled LNA miRNA probes on paraffin embedded heart sections. Next, we describe how ...

micro-RNAs (miRNAs) are short (18&#8211;25 nucleotides), single-stranded, noncoding RNAs that function as negative regulators of gene expression at the post-transcriptional level by inhibiting messenger RNA (mRNA) translation and/or promoting mRNA degradation1. miRNAs are transcribed from introns or exons of coding or noncoding genes and are cleaved in the nucleus by DROSHA, to precursor miRNAs (pre-miRNAs), which are short stem-loop structures of 70 nucleotides2. Following cytoplasmic export, pre-miRNAs are further processed by DICER into mature miRNAs that span 18&#8211;25 nucleotides3,4. Subsequently, the RNA-induced silencing complex (RISC) incorporates these miRNAs as single-stranded RNAs, which allows for their binding to the 3&#39; untranslated region (3&#39;-UTR) of their target mRNAs to suppress their expression3,5.
Within the last three decades, since they were first identified, miRNAs have emerged to master&#160;regulators of gene expression, whose own expression levels are tightly controlled6. Roles for miRNAs have been described in organ development7,8,9,10,11,12, maintenance of homeostasis13,14, as well as disease contexts that include neurological15,16,17,18,19, cardiovascular20, autoimmune conditions21,22, cancers23,24, and others25. The increasing appreciation for the relevance of miRNA expression patterns has brought forward the need for reliable detection methods of miRNA transcripts. Such methods include Real Time PCR, microarrays, Northern Blotting, in situ hybridization and others, which vary in the sensitivity, specificity, and quantitative power, predominantly due to the fact that miRNA transcripts are comprised of short and highly homologous sequences6.
We recently reported an important role for miRNA-182 in the development of the myocardial hypertrophy26, a condition describing the structural adaptation of the heart in response to elevated hemodynamic demands27,28. Cardiac hypertrophy is characterized by the increase in the myocardial mass, which, if associated with maladaptive remodeling29, can lead to increased risk for heart failure, a condition accounting for 8.5% of all deaths attributable to cardiovascular disease30.
Here, we describe our protocol that combines in situ hybridization with a digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe and immunostaining for the concurrent detection of miRNA and protein molecules on mouse heart tissue sections, in our model of cardiac hypertrophy.

<table>
	<tbody>
		<tr>
			<td>Diethylpyrocarbonate</td>
			<td>Sigma Aldrich</td>
			<td>D5758</td>
			<td>DEPC</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>P4417</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>American Bioanalytical</td>
			<td>AB02038</td>
			<td>non-ionic surfactant</td>
		</tr>
		<tr>
			<td>Histoclear</td>
			<td>National Diagnostics</td>
			<td>HS-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K, recombinant, PCR Grade</td>
			<td>Sigma Aldrich</td>
			<td>3115879001</td>
			<td>ProK</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td>PFA</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>ThermoFisher</td>
			<td>S271</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Hexahydrate</td>
			<td>ThermoFisher</td>
			<td>M33</td>
			<td>MgCl2</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma Aldrich</td>
			<td>T6066</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid Solution, 1 N</td>
			<td>ThermoFisher</td>
			<td>SA48</td>
			<td>HCl</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid Solution, 12 N</td>
			<td>ThermoFisher</td>
			<td>S25358</td>
			<td>HCl</td>
		</tr>
		<tr>
			<td>1-Methylimidazole</td>
			<td>Sigma Aldrich</td>
			<td>336092</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>39391</td>
			<td>EDC</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution H2O2</td>
			<td>Sigma Aldrich</td>
			<td>216763</td>
			<td>H2O2</td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>S1804</td>
			<td>Sodium Citrate</td>
		</tr>
		<tr>
			<td>miRCURY LNA miRNA ISH Buffer Set (FFPE)</td>
			<td>Qiagen</td>
			<td>339450</td>
			<td>scramble miRNA/U6 snRNA</td>
		</tr>
		<tr>
			<td>miRCURY LNA mmu-miR-182 detection probe</td>
			<td>QIagen</td>
			<td>YD00615701</td>
			<td>5&#39;-DIG and 3&#39;-DIG labelled</td>
		</tr>
		<tr>
			<td>Levamisol hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>31742</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A9647</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>NBT/BCIP Tablets</td>
			<td>Sigma Aldrich</td>
			<td>11697471001</td>
			<td>NBT-BCIP</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>ThermoFisher</td>
			<td>P217</td>
			<td>KCl</td>
		</tr>
		<tr>
			<td>DAPI solution (1mg/ml)</td>
			<td>ThermoFisher</td>
			<td>62248</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>ThermoFisher</td>
			<td>12-545E</td>
			<td>Glass coverslip</td>
		</tr>
		<tr>
			<td>Plastic coverslip</td>
			<td>Grace Bio-Labs</td>
			<td>HS40 22mmX40mmX0.25mm</td>
			<td>RNA-ase free plastic coverslip</td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin-AP, Fab fragments</td>
			<td>Sigma Aldrich</td>
			<td>11093274910</td>
			<td>DIG antibody</td>
		</tr>
		<tr>
			<td>Hydrophobic barrier pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td>Pap pen</td>
		</tr>
		<tr>
			<td>Anti-Cardiac Troponin T antibody</td>
			<td>Abcam</td>
			<td>ab92546</td>
			<td>cTnT antibody</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Absorbed Secondary Antibody, Alexa Fluor 568</td>
			<td>ThermoFisher</td>
			<td>A-11011</td>
			<td>anti-rabbit-568 antibody</td>
		</tr>
		<tr>
			<td>Dako Fluorescence Mounting Medium</td>
			<td>DAKO</td>
			<td>S3023</td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>Sheep serum</td>
			<td>Sigma Aldrich</td>
			<td>S3772</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized Formamide</td>
			<td>American Bioanalytical</td>
			<td>AB00600</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Oven</td>
			<td>ThermoFisher</td>
			<td>UVP HB-1000 Hybridizer</td>
			<td></td>
		</tr>
	</tbody>
</table>

tissue-specific mirna expression profiling in mouse heart sections using in situ hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

miRNA in situ hybridization was optimized on mouse heart sections using a scramble miRNA and U6-snRNA, which served as negative and positive controls respectively. Positive staining is indicated in blue, while the negative staining is indicated by the lack of color development (Figure 1A-1B). Cardiomyocyte specific expression of miRNA-182 was assessed in heart sections from control and PlGF overexpressing mice. The mice carrying the ...

Watch this Scientific Journal Video about Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization at JoVE.com

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

This method can help answer key questions in the microRNA field, by providing tools to detect the relative lcalization of both protein and microRNA molecules. The main advantage of this technique is that you can detect both protein and microRNA molecules from the same tissue section. This protocol begins with a rehydration of the slide-mounted tissues.Warm the slides at 60 degrees Celsius for 10 minutes in the hybridization oven. The paraffin should be visibly melting. Then, incubate the slides in a glass Coplin jar containing commercially-available clearing agent for 10 minutes.Do this twice. Next, transfer the slides from wash to wash to rehydrate the tissues. Once hydrated, incubate the tissues in proteinase K working solution at 37 degrees Celsius for 15 minutes.To remove the proteinase K, wash the slides in 1x PBS two times. Now, fix the tissues. First, make reservoirs on the slides using a hydrophobic pen to draw a water-repellent circle around the preparations.Next, apply 500 microliters of 4%PFA solution within the barrier, and incubate the slides in a fume hood. To remove the fixative, wash the slides in a Coplin jar containing PBS for five minutes two times at room temperature. Next, wash the slides in 0.13 molar 1-Methylimidazole for 10 minutes.Do this in the fume hood at room temperature, and perform this wash twice. Add the EDC fixative, and incubate at room temperature in a fume hood for one hour. Rinse off the slides with 50-millimolar Tris.To prepare for the hybridization, first build a humidified chamber. Load two pieces of filter or tissue paper into the bottom of a box, and wet the paper with a one-to-one mixture of formamide and 1x SSC. Next, apply 200 microliters of pre-warmed hybridization solution to each slide, and incubate the humidified chamber of slides for one to three hours at 50 degrees Celsius.After the incubation, replace the hybridization solution with 250 microliters of probe solution, and cover the slides with an RNase-free cover slip. Try to avoid trapping air bubbles under the cover slip. Now, carefully seal the chamber with parafilm, and incubate the slides overnight at approximately 30 degrees Celsius below the RNA melting temperature of the probe.Optimization may be required. In addition to setting the right incubation temperature, different microRNAs are expressed at different levels, so optimization regarding the probe concentration may also be required to get the optimal signal. Following the hybridization, perform the stringency washes.First, was in 2x SSC at 50 degrees Celsius for five minutes, or until the cover slips loosen. Then, perform two washes in 2x SSC at room temperature, each for five minutes. Then, continuing at room temperature, wash the slides for five minutes in 0.2x SSC, followed by five minutes in PBST, and finally, five minutes in 1x PBS.At this point, the RNA-RNA hybrids are stable, and RNA-stringent conditions are no longer needed. For DIG antibody detection, first, touch up the hydrophobic barriers, and apply 500 microliters of blocking solution for thirty minutes. Incubate the slides at room temperature in a humidified chamber.Next, remove the blocking solution and replace it with 500 microliters of DIG antibody solution. Incubate the slides at room temperature in a humidified chamber for an hour. Then, using Coplin jars, wash the slides three times in PBST for five minutes per wash.Next, wash the slides in pre-staining solution for five minutes twice. Then, transfer the slides to a polypropylene slide mailer jar, and add 20 milliliters of AP substrate solution. Now, incubate the slides at 37 degrees Celsius for six to 24 hours, protected from the light.When first using AP substrate, it is important to follow the reaction. On two-hour intervals, check the color development on the slides using a microscope, until an optimal incubation time has been determined. To remove the excess AP substrate, wash the slides twice in KTBT solution for five minutes per wash, followed by one wash in PBS for five minutes.To detect the cTNT antibody, first, block the tissues for an hour. Then, apply 200 microliters of cTNT antibody solution, and incubate the slides overnight at 4 degrees Celsius in a humidified chamber. The next day, wash the slides in a Coplin jar containing PBS for five minutes.Do this three times. Then, apply 250 microliters of anti-rabbit 568 antibody solution, and incubate the slides for an hour at room temperature in a humidified chamber. To wash out the excess secondary antibody, use three five-minute washes in PBS.Then, if desired, apply 250 microliters of DAPI working solution, and incubate the slides for one minute, protected from light. To remove the excess DAPI stain, use two five-minute PBS washes, and after the washes, mount the slides. MicroRNA in-situ hybridization was optimized on mouse heart sections using a scrambled microRNA for negative control, and a U6 snRNA as a positive control.Cardiomyocyte-specific expression of microRNA-182 was assessed in heart sections from control and PlGF-overexpressing mice. The mice carried a PlGF transgene under an alpha-MHC promoter, and developed cardiac hypertrophy, secondary to increased angiogenesis, within six weeks of transgene activation. The staining protocol revealed increased expression of microRNA-182 in the hearts of PlGF mice.Next, to determine which cell types expressed microRNA-182, the same sections were immunostained for cTNT. DAPI staining was also used. In both control and PlGF mouse heart sections, microRNA-182 was found in the nuclear compartment of cTNT-positive cardiomyocyte cells.After watching this video, you should have a good understanding of how to perform microRNAse hybridization, followed by protein immunostaining. While attempting this procedure, it is very important to remember to keep RNAse-free conditions throughout day one, during all steps leading up to prop hybridization. Following this procedure, other methods like Western blot in a real-time PCR can be performed to answer questions like, what are the relative expression levels of this particular microRNA in different tissues, or this particular protein in the same tissue section.Don't forget that working with fixatives such as PFA and ADC can be extremely hazardous. Precautions, include wearing gloves and lab coats, as well as using fume hoods, should always be taken when performing this procedure.

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JoVE Journal

Genetics

8.0K vistas.  University College London. Este método puede ayudar a responder preguntas clave en el campo del microARN, proporcionando herramientas para detectar la relativa lcalización de moléculas de proteínas y microARN. La principal ventaja de esta técnica es que se pueden detectar moléculas de proteínas y microARN de la misma sección tisular. Este protocolo comienza con una rehidratación de los tejidos montados en las diapositivas.Caliente los portaobjetos a 60 grados centígrados durante 10 minutos en el horno ...