Name: MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues (Video) | JoVE Journal
Uploaded: 2013-11-30T19:30:00.000+00:00
Duration: 12 min 21 s
Description: Learn in situ hybridization for microRNA detection in formalin-fixed kidney tissues using colorimetric visualization. This protocol enables precise localization and quantification of microRNA expression patterns.

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5 minutes

This work was supported by US National Institutes of Health grants HL082798 and HL111580.

1. Kidney Tissue Sections
<ol>
	<li>Formalin-fixed (10%) paraffin-embedded kidney sections are cut onto microscope slides at 5 mm thickness using a Microm HM 355 S. The slides can be stored for several months before analysis.</li>
</ol>
2. Solution Preparation
<ol>
	<li>Prepare 1 L of 1x PBS in ddH2O in an autoclavable screw top bottle. Fill another bottle with 1 L of ddH2O. Add 1 ml of DEPC to each bottle, cover and shake vigorously. Let the solutions sit overnight at room temperature to destroy RNAses and then autoclave. Add 400 ml Tween-20 to 1 L of 1x PBS to make PBS-T.</li>
	<li>Prepare proteinase K buffer (50 mMTris-HCl, pH 8.0 and 1 mM CaCl2 in DEPC treated water).</li>
	<li>Prepare a solution of 0.2% glycine in PBS-T.</li>
</ol>
3. Tissue Preparation
<ol>
	<li>Prepare a large volume of hybridization buffer (50-100 ml) composed of 50% Formamide, 5x SSC, 0.1% Tween, 9.2 mM citric acid for adjustment to pH 6, 50 &#181;g/ml heparin, and 500 &#181;g/ml yeast RNA in DEPC treated ddH2O. Divide into 1 ml aliquots and store at -80 &#186;C.</li>
	<li>Remove the paraffin from the tissue by washing 3x in fresh xylene for 5 min each.</li>
	<li>Rehydrate the tissue sections by 5 min incubations in decreasing concentrations of ethanol (100%, 75%, 50%, and 25%) in ddH2O.</li>
	<li>Treat the slides with enough DEPC to completely cover the tissue sections (approximately 0.4-0.5 ml) for 1 min. Make sure tissues do not dry out.</li>
	<li>Rinse the slides in DEPC treated PBS-T twice for 5 min, collecting the DEPC containing waste for proper disposal. All reagents should be DEPC treated to eliminate RNAses from this point on.</li>
	<li>Prewarm Proteinase K buffer and add proteinase K to a final concentration of 10 &#956;g/ml. Cover tissue sections with proteinase K solution and incubate at 37 &#186;C for 5 min.</li>
	<li>Quickly remove the proteinase K solution and add 0.2% glycine in PBS-T for 30 sec.</li>
	<li>Rinse slides in DEPC treated PBS-T twice for 30 sec each.</li>
	<li>Fix sections in freshly made 4% paraformaldehyde for 10 min.</li>
</ol>
4. Hybridization
<ol>
	<li>Add 50 &#181;l of hybridization buffer (50% Formamide, 5x SSC, 0.1% Tween, 9.2 mM citric acid for adjustment to pH 6, 50 &#181;g/ml heparin, 500 &#181;g/ml yeast RNA) per tissue section and cover with RNAse free HybriSlips cut just larger than the tissue sections.</li>
	<li>Prehybridize sections in humidity controlled hybridization oven for 2 hr at hybridization temperature determined for the 3&#8217; digoxigenin labeled probe (usually DNA Tm of the probe -21 &#176;C, ( i.e.&#160;54 &#176;C for miR-382, probe DNA Tm= 75 &#176;C), using tissue lab wipes soaked in 50% formamide/50% 5x SSC to keep the chamber humidified.</li>
	<li>Dilute the miRNA probe, positive control (U6, small nuclear RNA) and negative control (scrambled sequence) to 40 nM in hybridization buffer (75 &#181;l of buffer is needed per section).</li>
	<li>Heat the probe in hybridization buffer at 65&#160;&#186;C for 5 min.</li>
	<li>Remove slides from hybridization oven and remove coverslips and excess hybridization buffer from prehybridization.</li>
	<li>Add 75 &#181;l of probe containing buffer per tissue section, directly to tissue. Cover tissue with HybriSlips just large enough to cover the tissue sections.</li>
	<li>Keeping slide holder level, return to hybridization oven for overnight incubation at temperature from step 4.2 (approximately 16 hr).</li>
</ol>
5. Stringency Wash
<ol>
	<li>Add 200 &#181;l 2x SSC, heated to the hybridization temperature, just under one edge of the coverslips to help release the coverslip from the tissue. Remove the coverslip by tilting the slide.</li>
	<li>Wash the slides in 200 &#181;l of 50% formamide, 50% 2x SSC at hybridization temperature 3x for 30 min each.</li>
	<li>Rinse the slides 5x in PBS-T at room temperature for 5 min each on orbital shaker on low speed.</li>
</ol>
6. Detection
<ol>
	<li>Incubate slide in blocking buffer (2% goat or horse serum, 2 mg/ml BSA in PBS-T) for 1 hr at room temperature on orbital shaker on low speed.</li>
	<li>Dilute anti-DIG-AP Fab fragments in blocking buffer at 1:100. Add 100 &#181;l per tissue section and cover with HybriSlips cut just large enough to cover the section.</li>
	<li>Incubate antibody in a humidified chamber at 4 &#176;C overnight (approximately 16 hr).</li>
	<li>Wash the slides in PBS-T 7x, for 5 min each on orbital shaker at low speed.</li>
	<li>Wash the slides in AP buffer (100 mM TrisHCl pH 9.5, 50 mM MgCl2,100mM NaCl, 0.1% Tween 20) 3x, for 5 min each.</li>
	<li>Add NBT/BCIP solution to AP buffer (200 &#181;l/10 ml buffer)</li>
	<li>Add 400-500 ml of diluted NBT/BCIP solution to each slide to completely cover all tissue sections. Develop in a dark, level, humidified chamber for 4-5 hr.</li>
</ol>
7. Mounting Slides
<ol>
	<li>Dehydrate sections in increasing concentrations of ethanol (25%, 50%, 75%, 100%) for 5 min each.</li>
	<li>Incubate in xylene 5x in order to clear sections.</li>
	<li>Mount slides in Permount mounting medium and dry overnight.</li>
</ol>

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<li>Kato, M., Arce, L., Wang, M., Putta, S., Lanting, L., Natarajan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+microRNA+circuit+mediates+transforming+growth+factor-%CE%B21+autoregulation+in+renal+glomerular+mesangial+cells%5BTitle%5D)+AND+%22Kidney+Int%22%5BJournal%5D)">A microRNA circuit mediates transforming growth factor-β1 autoregulation in renal glomerular mesangial cells.</a> Kidney Int. 80 (4), 358-368 (2011).</li>
<li>Gottardo, F., Liu, C. G., Ferracin, M., Calin, G. A., Fassan, M., Bassi, P., Sevignani, C., Byrne, D., Negrini, M., Pagano, F., Gomella, L. G., Croce, C. M., Baffa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micro-RNA+profiling+in+kidney+and+bladder+cancers%5BTitle%5D)+AND+%22Urol.+Oncol%22%5BJournal%5D)">Micro-RNA profiling in kidney and bladder cancers.</a> Urol. Oncol. 25 (5), 387-392 (2007).</li>
<li>Juan, D., Alexe, G., Antes, T., Liu, H., Madabhushi, A., Delisi, C., Ganesan, S., Bhanot, G., Liou, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+a+microRNA+panel+for+clear-cell+kidney+cancer%5BTitle%5D)+AND+%22Urol%22%5BJournal%5D)">Identification of a microRNA panel for clear-cell kidney cancer.</a> Urol. 75 (4), 835-841 (2010).</li>
<li>Xu, X., Kriegel, A. J., Liu, Y., Usa, K., Mladinov, D., Liu, H., Fang, Y., Ding, X., Liang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delayed+ischemic+preconditioning+contributes+to+renal+protection+by+upregulation+of+miR-21%5BTitle%5D)+AND+%22Kidney+Int%22%5BJournal%5D)">Delayed ischemic preconditioning contributes to renal protection by upregulation of miR-21.</a> Kidney Int. 82 (11), 1167-1175 (2012).</li>
<li>Kriegel, A. J., Mladinov, D., Liang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translational+study+of+microRNAs+and+its+application+in+kidney+disease+and+hypertension%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Translational study of microRNAs and its application in kidney disease and hypertension.</a> 122 (10), 439-447 (2012).</li>
<li>Kloosterman, W. P., Wienholds, E., de Bruijn, E., Kauppinen, S., Plasterk, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+detection+of+miRNAs+in+animal+embryos+using+LNA-moidified+oligonucleotide+probes%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">In situ detection of miRNAs in animal embryos using LNA-moidified oligonucleotide probes.</a> Nat. Methods. 3 (1), 27-29 (2006).</li>
<li>Nelson, P. T., Baldwin, D. A., Kloosterman, W. P., Kauppinen, S., Plasterk, R. H., Mourelatos, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RAKE+and+LNA-ISH+reveal+microRNA+expression+and+localization+in+archival+human+brain+tissue%5BTitle%5D)+AND+%22RNA%22%5BJournal%5D)">RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain tissue.</a> RNA. 12 (2), 187-191 (2006).</li>
<li>Obernosterer, G., Martinez, J., Alenius, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Locked+nucleic+acid-based+in+situ+detection+of+microRNAs+in+mouse+tissue+sections%5BTitle%5D)+AND+%22Nat.+Protocols%22%5BJournal%5D)">Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections.</a> Nat. Protocols. 2 (6), 1508-1514 (2007).</li>
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There is nothing to disclose.

The goal of this article was to describe a protocol for miRNA in situ hybridization that works well in formalin fixed kidney tissues. While working out this protocol several important sources of staining artifact have been identified. Careful attention to these points can help avoid staining artifact and increase the likelihood of a successful ISH run.
One of the most avoidable causes of staining artifact can occur when tissue sections become dried out during long incubations. When th...

MicroRNA are small (approximately 22 nucleotides long) noncoding RNAs that are endogenously produced. They generally function to suppress protein expression through translational repression or mRNA degradation. miRNAs bind to mRNA targets with incomplete complementarity, making it possible for a single miRNA to suppress multiple targets.
Understanding which cell types and structures express miRNAs is an important part of understanding the mechanisms through which alterations in miRNA expression influence cell and tissue phenotypes. While methods such as miRNA sequencing, qPCR and Northern blotting can be used for detection of miRNAs in whole tissues, this approach does not allow one to determine which specific cell type they came from within a given tissue. Dissection of cellular and structural components prior to analysis using these methods can be very difficult and the conditions needed to achieve adequate isolations can lead to alterations in gene expression or degradation of RNAs. microRNA in situ hybridization is a method used to visualize microRNA location and expression levels in tissues. This technique is especially valuable in tissues composed of heterogeneous structures, such as the kidney.
MicroRNAs have been shown to play an regulatory role in kidney development2,3 and physiology4. microRNA expression alterations have also been shown to be involved with renal pathology such as fibrosis5-10, diabetic nephropathy7, renal carcinoma11,12, and acute kidney injury13. In our research, we have found that optimizing microRNA in situ hybridization for kidney tissues was valuable for determining the exact structural locations of miRNA expression in both health and disease14. Determination of the tubular and cellular expression of different microRNAs is important because their regulation of targets may be dependent on cellular functions. In diseased states it is also important to determine how alterations in miRNA expression may be impacting function.
The goal of the method described here was to build upon existing ISH methodologies developed by Kloosterman et al.15, other investigators16,17, and those suggested by Exiqon1 and optimize the method for formalin fixed kidney tissues. We have successfully used this method to identify distinct regional differences in renal microRNA miR-382 expression with unilateral ureteral obstruction18. This approach can be used with other tissues as well, with additional optimization.

<table><tbody><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>70011044</td><td></td></tr><tr><td>Diethyl Pyrocarbonate</td><td>Sigma</td><td>D5758</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>Xylenes</td><td>Sigma</td><td>534056</td><td></td></tr><tr><td>Ethanol</td><td>Ultra Pure</td><td>200CSPTP</td><td></td></tr><tr><td>Tris-HCl</td><td>Life Technologies</td><td>15506-017</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C2536</td><td></td></tr><tr><td>Glycine</td><td>J.T. Baker</td><td>4059-00</td><td></td></tr><tr><td>Citric Acid</td><td>Sigma</td><td>C2404</td><td></td></tr><tr><td>Formamide</td><td>Sigma</td><td>F9037</td><td></td></tr><tr><td>20x SSC Buffer</td><td>Life Technologies</td><td>15557-044</td><td></td></tr><tr><td>Heparin</td><td>SAGENT</td><td>25021-400-30</td><td></td></tr><tr><td>Yeast RNA</td><td>Life Technologies</td><td>AM7118</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266-1004</td><td></td></tr><tr><td>NaCl</td><td>J.T. Baker</td><td>4058-01</td><td></td></tr><tr><td>Proteinase K</td><td>Fermentas</td><td>EO0491</td><td></td></tr><tr><td>3&amp;rsquo; DIG- miRNA probe</td><td>Exiqon</td><td>miR dependent-05</td><td></td></tr><tr><td>3&amp;rsquo; DIG- Scrambled miR probe</td><td>Exiqon</td><td>99004-05</td><td></td></tr><tr><td>3&amp;rsquo; DIG- U6 miR probe</td><td>Exiqon</td><td>99002-05</td><td></td></tr><tr><td>Anti-Digoxigenin-Ab Fab</td><td>Roche</td><td>11093274910</td><td></td></tr><tr><td>NBT/BCIP</td><td>Roche</td><td>11681451001</td><td></td></tr><tr><td>Permount</td><td>Fisher</td><td>SP15-500</td><td></td></tr><tr><td>Coverslips</td><td>Fisher</td><td>Fit to tissue size</td><td></td></tr><tr><td>Microtom HM 355 S</td><td>Thermo Scientific</td><td>23-900-672</td><td></td></tr><tr><td>In-slide Out Hybridization Oven</td><td>Boekel</td><td>241000</td><td></td></tr><tr><td>Aluminum Tray Assembly</td><td>Boekel</td><td>C2403973</td><td></td></tr><tr><td>Stainless Steel Rack Insert</td><td>Boekel</td><td>C2403754</td><td></td></tr></tbody></table>

microrna in situ hybridization for formalin fixed kidney tissues

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

The areas of a tissue section that become dried out during the hybridizations, incubations or wash steps generally end up staining more darkly during the NBT/BCIP development. Figure 1 shows a portion of a kidney section in which the HybriSlip slipped off of the edge of the tissue, allowing it to become partially dehydrated. Despite rehydration and coverage in the remaining steps, the signal in the dehydrated portion is artificially high.
The importance of controlling the time...

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

The overall goal of the following experiment is to locate and quantify micro RNA expression within kidney tissues. This is achieved by first hybridizing, a diggen and tagged probe to a given micro RNA within the kidney tissue. As a second step, an antibody to digo genin, which has a conjugated alkaline phosphatase, is incubated with the tissue.Next, an N-B-T-B-C-I-P substrate is added to the tissue in order to visualize the location of the micro RNA through development of a blue colored product at sites of the hybridized micro RNA probes results are obtained that show where microRNAs are expressed in kidney regions and nephron segments based on hybridization of micro RNA complimentary probes. This procedure uses formal and fixed paraffin embedded kidney sections that are caught onto microscope slides at a thickness of five microns. To prepare the kidney sections for analysis, first remove the paraffin from the tissue by three washes in fresh xylene for five minutes.Each rehydrate the tissue sections by five minute incubations in decreasing concentrations of ethanol in doubly distilled water. Next, treat the slides with enough dathyl, percarbonate, or desy to completely cover the tissue sections for one minute. Make sure the tissues do not dry out.Remove the desy and rinse the slides in. Dessy treated PBST twice for five minutes. Make sure the dessy containing waste is collected for proper disposal.From this point on, all reagents should be dsy treated To eliminate RNAs, add proteinase K to prewarm proteinase K buffer to a final concentration of 10 micrograms per milliliter. Cover the tissue sections with the protein A K solution and incubate at 37 degrees Celsius for five minutes. Quickly remove the proteinase K solution and add 0.2%glycine in PBST for 30 seconds.Rinse the slides in. Desy treated PBST twice for 30 seconds each. Lastly, fix the tissue sections in freshly made 4%para formaldehyde for 10 minutes.To begin this procedure, add 50 microliters of hybridization buffer per tissue section and cover with RNAs free hybrid slips that are caught just larger than the tissue sections. Place the slides inside a chamber humidified by lab wipes soaked in 50%form amide 50%five XSSC pre hybridized the tissue sections by placing the humidified chamber for two hours in an oven at the hybridization temperature determined for the three prime dig oxygen and labeled probe. The hybridization temperature for the probe is usually the DNA melting temperature or TM of the probe minus 21 degrees Celsius.For example, the hybridization temperature for the mere 3 82 probe is 54 degrees Celsius because its TM is 75 degrees Celsius. Prepare the micro RNA probe and the positive and negative controls by diluting each to 40 nano molar in hybridization buffer. The positive control is the U six small nuclear RNA and the negative control is a scrambled sequence.Heat the probe and the controls at 65 degrees Celsius for five minutes. Remove the slides from the hybridization oven. Remove the hybrid slips and excess hybridization buffer from the pre hybridization setup.Add 75 microliters of probe containing buffer directly onto each tissue section. Cover with hybrid slips that are, again, just large enough to cover the tissue sections. Keeping the slide holder level.Return the slides to the hybridization oven at the appropriate temperature. Incubate overnight for approximately 16 hours. Once hybridization is complete, remove the slides from the hybridization oven.Add 200 microliters of two XSSC heated to the hybridization temperature just under one edge of the cover slip to help release the hyper slip from the tissue. Remove the hyper slip by tilting the slide. After the cover slips have been removed, wash the slides in 200 microliters of 50 percentfor amide 50%two XSSC at the determined hybridization Temperature three times for 30 minutes each.Rinse the slides five times in PBST at room temperature for five minutes each on an orbital shaker at low speed. Begin this procedure by incubating the slides in blocking buffer for one hour at room temperature on an orbital shaker at low speed. After one hour, add 100 microliters of anti dig AP FAB fragments to each tissue section cover with hybrid slips.Cut just large enough to cover the section. Incubate the slides in a humidified chamber at four degrees Celsius overnight or for approximately 16 hours the following day. Wash the slides seven times in PBST at room temperature for five minutes each time on an orbital shaker set at low speed.Next, wash the slides in AP buffer three times for five minutes each. Prepare the N-B-T-B-C-I-P developing solution by adding 200 microliters of N-B-T-B-C-I-P solution to 10 milliliters of AP buffer. Add 400 to 500 microliters of the diluted N-B-T-B-C-I-P solution to each slide to completely cover all tissue sections.Develop in a dark level humidified chamber for four to five hours at room temperature when the chole metric detection is complete. Dehydrate the sections in increasing concentrations of ethanol for five minutes each clear the sections through five incubations in xylene for five minutes each time. Lastly, mount the slides in per mount mounting medium and dry them overnight.Ensuring that the tissue section does not dry out is critical to the success of this insi hybridization protocol. The left part of this figure shows a portion of a kidney section in which the hybrid slip slipped off of the edge of the tissue, allowing it to become partially dehydrated. Despite rehydration and coverage in the remaining steps, the signal in the dehydrated portion is artificially high compared to the portion that remained covered by the hyper slip.The calibration bar represents 100 microns in this image. The importance of optimizing the duration of N-B-T-B-C-I-P development is demonstrated in these next figures. Panel A shows that development periods of longer than four to five hours results in pronounced nonspecific color development and scrambled micro RNA control probed tissues throughout the kidney.Panel B shows that incubations longer than four to five hours do not result in further improvement in signal when kidneys are probed for high abundance targets such as the U six small nuclear RNA control. These figures adapted from a previous study confirm the applicability of this in situ hybridization method to micro RNA detection in kidney tissue panel A shows representative images of mere 3 82 expression obtained by insi hybridization in kidney sections from mice with or without unilateral ureteral obstruction or UUO and treated with either anti-ME 3 82 or antis. Scrambled UUO induced an increase in mere 3 82 expression as shown by the darker staining in the UUO plus antis scrambled tissue compared to the sham plus antis scrambled tissue.This increase was blocked by treatment with anti-ME 3 82. The graphs in panel B show mere 3 82 expression of sham plus antis scrambled treated mice as percent average integrated optical density. A measure of pixel intensity as observed, UUO induces a significant increase in mere 3 82 expression in the outer and inner medulla and anti-ME 3 82 blocks.This increase, all of which can be visualized and measured by micro RNA in situ hybridization. These results are also consistent with results from Q PCR R that are not shown here, indicating that the use of INI two hybridization to detect mere 3 82 expression in formal and fixed kidney is sensitive enough to provide quantifiable results that mirrored qPCR R based expression analysis. Additionally, in C two hybridization allows the measurement of distinct regional differences in micro RNA expression within the kidney.

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Research

JoVE Journal

Biology

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections.  The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

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

We describe a protocol for laser microdissection of sub-segments of the human kidney, including the glomerulus, proximal tubule, thick ascending limb, collecting duct and interstitium. The RNA is then isolated from the obtained compartments and RNA sequencing is carried out to determine changes in the transcriptomic signature within each sub-segment.

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue

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A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction

microRNAs are involved in the pathogenesis of IgA nephropathy. We have developed a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). This new method will facilitate to check for miRNAs involvement in IgA nephropathy.

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

The protocol describes a tandem immunofluorescence hybridization assay with formalin-fixed paraffin-embedded tissue to detect and quantify microRNA and multiplexed proteins simultaneously.

MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as a Novel Detection and Quantification Method

Learn in situ hybridization for microRNA detection in formalin-fixed kidney tissues using colorimetric visualization. This protocol enables precise localization and quantification of microRNA expression patterns.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

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