We thank Michelle Goody, PhD, from Edanz Group (https://en-author-services.edanzgroup.com/) for editing a draft of this manuscript.

All animal experimental protocols were approved by the Animal Ethics Committee of Jichi Medical University and were performed in accordance with the Use and Care of Experimental Animals guidelines from the Jichi Medical University Guide for Laboratory Animals.
1. The sham surgery
<ol>
	<li>Prepare the following items: isoflurane, cork sheet, depilatory cream, laboratory wipes, Petri dish with phosphate-buffered saline (PBS), 4-0 nylon, tweezers, surgical scissors, cotton swabs, and 8-week-old C57BL/6 male mice.</li>
	<li>Anesthetize a mouse with 1.5% isoflurane and maintain at 1.5%. Then, apply depilatory cream to the mouse&#39;s abdomen. After a few minutes, wipe the depilatory cream off with a PBS-soaked laboratory wipe.</li>
	<li>Inject 70% ethanol into the mouse&#39;s abdomen and then place the mouse on the cork sheet in the supine position.</li>
	<li>Using surgical scissors and tweezers, make an incision in the skin at the abdomen and cut the muscle and peritoneal membrane from the bladder to the left lower edge of the ribs.</li>
	<li>Moisten two cotton swabs with PBS and then pull the intestines carefully to the side. Place the moistened swabs to identify the left kidney and ureter.</li>
	<li>Close the peritoneal membrane and then close the incision with 4-0 nylon.</li>
</ol>
2. The UUO surgery
<ol>
	<li>Prepare the following items: isoflurane, cork sheet, depilatory cream, laboratory wipes, Petri dish with PBS, 4-0 silk, 4-0 nylon, a 2.5 mL syringe, cotton swabs, tweezers, surgical scissors, and 8-week-old C57BL/6 male mice.</li>
	<li>Anesthetize a mouse with 1.5% isoflurane and maintain at 1.5%. Then apply depilatory cream to the mouse&#39;s abdomen. After a few minutes, wipe the depilatory cream off with a PBS-soaked laboratory wipe.</li>
	<li>Inject 70% ethanol into the mouse&#39;s abdomen mouse and then place the mouse in the supine position on the cork sheet.</li>
	<li>Using surgical scissors and tweezers, make an incision in the skin at the abdomen and cut the muscle and peritoneal membrane from the bladder to the left lower edge of the ribs.</li>
	<li>Place the 2.5 mL syringe underneath the mouse. Take two cotton swabs and moisten them with PBS. Pull the intestines carefully to the side with the tweezers, and place the moistened swabs appropriately to identify the left ureter. Using the tweezers, lift the left kidney.</li>
	<li>Use the 4-0 silk to ligate the left ureter in two places approx. 1 cm apart. Cut the ureter at the center point of the two ligations, and then use 4-0 nylon sutures to close the peritoneal membrane and incision.</li>
</ol>
3. Collection of kidney samples
<ol>
	<li>Prepare the following: 1.5 mL microcentrifuge tubes, isoflurane, cork sheet, 70% ethanol, Petri dish with PBS, tweezers, and surgical scissors.</li>
	<li>Anesthetize a mouse with 1.5% isoflurane and maintain at 1.5%. Inject 70% ethanol into its abdomen and put the mouse on the cork sheet in the supine position.</li>
	<li>Using surgical scissors and tweezers, make an incision in the skin at the abdomen and cut the muscle and peritoneal membrane from the bladder to the left lower edge of the ribs.</li>
	<li>Lift up the peritoneal membrane with the tweezers. With the surgical scissors, make a sideways incision at the upper edge of the peritoneal membrane, and continue the incision along the lowest edge of the ribs.</li>
	<li>Next, identify the left kidney, reflux it with PBS until the kidney turns yellow-white to wash out blood from vessels, and remove the kidney by cutting the left renal artery and vein with the surgical scissors. Place the kidney in the Petri dish and wash it carefully with PBS.</li>
	<li>Cut the kidney into 10 mg samples with the surgical scissors and tweezers (10 mg is an appropriate size for the next step). Put each piece of the kidney in its own 1.5 mL microcentrifuge tube and close the tube&#39;s cap.</li>
	<li>Transfer each microcentrifuge tube into liquid nitrogen, and keep the tubes at &#8722;80 &#176;C for long-term storage before use.</li>
</ol>
4. Extraction of total RNA from the kidney samples
<ol>
	<li>Prepare the following items: 1.5 mL microcentrifuge tubes, 2.0 mL microcentrifuge tubes, 100% ethanol, chloroform, silicon homogenizer, ice, a vortex mixer, biopolymer spin columns in 2.0 mL collection tubes11,12, membrane-anchored spin columns in 2.0 mL collection tubes12,13, phenol/guanidine-based lysis reagent, wash buffer containing guanidine and ethanol (wash buffer 1), wash buffer containing ethanol (wash buffer 2), and RNase-free water.</li>
	<li>Put a 10 mg kidney sample in the silicon homogenizer. Add 700 &#181;L of the phenol/guanidine-based lysis reagent in the homogenizer.</li>
	<li>Prepare the homogenizer and then slowly press/twist the homogenizer&#39;s pestle against the kidney sample to homogenize the sample. Repeat the pressing/twisting until the kidney sample is completely dissolved in the phenol/guanidine-based lysis reagent.</li>
	<li>To further homogenize the sample, transfer the homogenized lysate (in a 2.0 mL collection tube) to the biopolymer spin column.</li>
	<li>Spin the homogenized lysate at 14,000 x g for 3 min at room temperature (RT), and then transfer the precipitated lysate to an unused 1.5 mL microcentrifuge tube.</li>
	<li>Combine the lysate in the tube with 140 &#181;L of chloroform and then close the tube cap tightly. To mix the lysate and chloroform, invert the tube 15 times. 
	NOTE: The chloroform can be used without a hood.</li>
	<li>Incubate each sample for 2&#8211;3 min at RT and then spin each sample at 12,000 x g for 15 min at 4&#176;C.</li>
	<li>Without disturbing the precipitate, transfer the supernatant (which is typically ~300 &#181;L) to a new 1.5 mL microcentrifuge tube, and then add 1.5x its volume (typically ~450 &#181;L) of 100% ethanol. Vortex the mixture for 5 s.</li>
	<li>Load 700 &#181;L of the sample onto a membrane-anchored spin column in a 2.0 mL collection tube. Close the column cap and spin the column at 15,000 x g for 15 s. Throw away the precipitated lysate in the collection tube.</li>
	<li>Wash the sample thoroughly by adding 700 &#181;L of wash buffer 1 to the membrane-anchored spin column in a 2.0 mL collection tube. Close the column cap, and spin the column at 15,000 x g for 15 s. Throw away the precipitated lysate in the collection tube.</li>
	<li>Load 500 &#181;L of wash buffer 2 onto the membrane-anchored spin column in a 2.0 mL collection tube to remove trace salts. Close the column cap, and spin the column at 15,000 x g for 15 s. Throw away the precipitated lysate in the collection tube. 
	NOTE: A membrane-anchored spin column can separate RNA and DNA.</li>
	<li>Perform step 4.11 again.</li>
	<li>Spin the membrane-anchored spin column in a 2.0 mL collection tube again at 15,000 x g for 1 min. Throw away the precipitated lysate in the collection tube.</li>
	<li>Transfer the membrane-anchored spin column to a new 1.5 mL collection tube. Dissolve total RNA by adding 30 &#181;L of RNase-free water to the column. Close the column cap, and wait 5 min at room temperature. Then, spin the column at 15,000 x g for 1 min.</li>
	<li>Transfer the total amount of sample containing total RNA to a new microcentrifuge tube. Put each of the tubes on ice, and measure the concentration of total RNA by spectrophotometry.</li>
	<li>Keep the tubes with samples at &#8722;80 &#176;C for long-term storage before use.</li>
</ol>
5. Synthesis of cDNA with the reverse transcription of total RNA
NOTE: The MIQE (Minimum Information for the Publication of Quantitative Real-Time PCR Experiments) guidelines were issued to encourage better experimental practices and help obtain reliable and unequivocal results19. In this protocol, cDNA is synthesized from 1.0 &#181;g of purified total RNA in a two-step procedure using reverse transcriptase, poly(A) polymerase, and oligo-dT primer.
<ol>
	<li>Prepare the following: 1.5 mL microcentrifuge tubes, eight-well strip tubes with caps, the cap of each eight-strip tube, distilled water, ice, a reverse transcriptase kit (see the Table of Materials)14,15 in the melted state, a thermal cycler, and a vortex mixer.</li>
	<li>Start the thermal cycler.</li>
	<li>Prepare a master mix solution: Add 2.0 &#181;L of reverse transcriptase mix (included in the kit) and 2.0 &#181;L of 10x nucleic acid mix into 4.0 &#181;L of 5x hi-spec buffer (to obtain a total of 8.0 &#181;L master mix per tube).</li>
	<li>Put 8.0 &#181;L of the master mix solution into each tube of an eight-well strip tube.</li>
	<li>Adjust the total RNA density. To isolate 1.0 &#181;g of total RNA from the kidney samples in 12 &#181;L of RNase-free water, transfer the appropriate amount of total RNA into distilled water, using the density data measured as described in step 4.15. 
	NOTE: If any DNA contamination is present, the contaminated DNA will be co-amplified in the qRT-PCR.</li>
	<li>Place a 12 &#181;L aliquot of total RNA into each tube and close the tube&#39;s cap. Centrifuge the tube for 15 x.</li>
	<li>Put the tube in the thermal cycler and incubate the sample for 60 min at 37 &#176;C. Next, immediately incubate the sample for 5 min at 95 &#176;C for the synthesis of cDNA.</li>
	<li>When the incubation is complete, transfer the cDNA into a new 1.5 mL microcentrifuge tube and dilute the cDNA ten times (1:10) with distilled water. Vortex and centrifuge the tube for 5 s.</li>
	<li>Temporarily store the diluted cDNA on ice and move the samples to &#8722;80 &#176;C for long-term storage before use.</li>
</ol>
6. qRT-PCR of miRNA
NOTE: We used the intercalator method to perform the qRT-PCR of miRNA. Primers for RNA are U6 small nuclear 2 (RNU6-2), miRNA-3070-3p, miRNA-6401, miRNA-7218-5p, and miRNA-7219-5p were used.
<ol>
	<li>Prepare the following: 1.5 mL microcentrifuge tubes, a vortex mixer, a 96-well reaction plate for the qRT-PCR, adhesive film for the 96-well reaction plate, an adhesive film applicator, a 96-well centrifuge rotor, miRNA-specific primers, a real-time PCR instrument, and a green dye-based PCR kit (see the Table of Materials)14,15 containing 2x PCR master mix and 10x universal primer.</li>
	<li>After mixing them in 1.5 mL microcentrifuge tubes, vortex the following items: 6.25 &#181;L of distilled water, 1.25 &#181;L of each 5 &#181;M miRNA primer dissolved in nuclease-free water, 12.5 &#181;L of 2&#215; PCR master mix, and 2.5 &#181;L of 10x universal primer.</li>
	<li>Prepare the cDNA synthesized as described in step 5, and melt it. Vortex and centrifuge the cDNA for 5 s.</li>
	<li>Put a 22.5 &#181;L aliquot of the reagent (created as described in step 6.2) in each well of the 96-well plate.</li>
	<li>Put a 2.5 &#181;L aliquot of cDNA in each well of the plate.</li>
	<li>Using the adhesive film applicator, secure the adhesive film tightly on the 96-well plate. Centrifuge the plate with the 96-well centrifuge rotor at 1,000 x g for 30 s to settle the reactions at the bottom of each well.</li>
</ol>
7. PCR cycling
<ol>
	<li>Start the real-time PCR system, and place the plate created as described in step 6.6. in the real-time PCR system. Set the experiment properties next; identify the experiment name. Select the following: &#34;96-well (0.2 mL)&#34; as the system&#39;s experiment type, &#34;Comparative CT (&#916;&#916;CT)&#34; as the quantitation method, &#34;SYBR Green Reagents&#34; as the reagents to detect the target sequence, and &#34;standard&#34; as the system&#39;s run.</li>
	<li>Assign a name to the sample and the target miRNA, and then assign a name to the sample and target miRNA in each well. Samples should be assigned in duplicate in order to obtain appropriate data for confirmation of the results. Select a reference sample and an endogenous control, and select &#34;none&#34; for the dye to be used as the passive reference. Make sure to set the negative reverse transcriptase and non-template control for the miRNA expression in order to eliminate the cross-contamination of reagents.</li>
	<li>Make sure the reaction volume setting is &#34;20 &#181;L&#34; and the PCR cycling conditions are set at 95 &#176;C for 15 min, followed by 40 cycles of denaturation at 94 &#176;C for 15 s, annealing at 55 &#176;C for 30 s, and extension at 70 &#176;C for 30 s.</li>
	<li>After the qRT-PCR process is complete, use the system&#39;s software program to analyze the qRT-PCR data. Ensure that the threshold line that is automatically selected by the software program is appropriate for each well.</li>
	<li>Check the threshold cycle (CT) value of the endogenous control and target miRNA analyzed in each sample. The CT value is determined by the intersection of the amplification curve and the threshold line. In the present study, we used RNU6-2 as an endogenous control for the target miRNA expression level, and we used the &#916;&#916;CT method to determine the relative expression level of each target miRNA20.</li>
</ol>

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The authors declare that they have no conflicts of interest.

The above-described protocol with qRT-PCR successfully determined the expression levels of the targeted miRNAs. The assessment of extracted miRNAs is important when seeking to obtain meaningful qRT-PCR data, and in order to confirm the quality of the miRNAs before performing the qRT-PCR, the ratio of absorbance at 260 nm to that at 280 nm should be checked with a spectrophotometer. If a single PCR amplification of the expected length and melting temperature or a monomodal melting curve cannot be obtained by qRT-PCR, there may be DNA contamination or a primer dimer in each well of the reaction plate.
The expression levels of miRNAs can be evaluated by several methods other than qRT-PCR, including microarray, northern blotting, and ribonuclease protection assay methods. However, qRT-PCR is a simple, highly reproducible procedure that is both accurate and sensitive, and smaller sample volumes can be used for qRT-PCR compared to northern blotting and ribonuclease protection assays22. In addition, since microarrays enable the simultaneous measurement of the expression of tens of thousands of miRNAs, they can identify candidate miRNA markers. Microarray data have shown an overall high correlation with data obtained by qRT-PCR23, but a consensus on the optimal methodology for comparing the microarray data obtained in disparate studies has not been reached24.
The new protocol has the following limitations. The utility of this protocol has not been validated in other organs, such as liver and lung; and the protocol has not been tested in other laboratory animals (e.g., rats, dogs, and pigs). Several groups reported that this protocol (for the purification and detection of miRNAs by qRT-PCR) enabled the purification of high-quality RNA from tissues12,14,15. The method has high accuracy and sensitivity for detecting miRNA expression12,14. The present report demonstrates that this protocol can be used to successfully detect miRNA expression in mouse kidney. The protocol can thus be used to determine the miRNA expression profiles in the kidneys of mice with a wide range of disease states. Due to the protocol&#39;s simplicity, many samples can be processed simultaneously, and using the protocol can therefore contribute to the analyses of the expression of many miRNAs in various pathological conditions of the kidney.
There are certain aspects of the protocol to be aware of and keep in mind. First, the purified RNAs must be kept on ice to prevent degradation at room temperature. The kidney samples must be homogenized until the samples are completely melted in the lysis reagent. Since mouse kidneys contain substantial connective tissue that does not dissolve in lysis reagent, a column shredder is necessary for further homogenization. Second, the proper endogenous control miRNA (whose expression is stable among samples) must be verified throughout the qRT-PCR experimental setup because the invasion of various substances under this protocol could change the expression level of the endogenous control miRNA, possibly compromising the results.
In conclusion, we have presented the details of a qRT-PCR protocol for the detection, purification, and evaluation of microRNA expressions in mouse kidney with UUO.

MicroRNAs (miRNAs)&#8201;&#8212;&#8201;the short, noncoding RNAs that cause the degradation and transcriptional inhibition of messenger RNA (mRNA)1&#8201;&#8212;&#8201;have been shown to regulate the expression of various mRNAs that have crucial roles in both physiology and disease (e.g., inflammation, fibrosis, metabolic disorders, and cancer). Some of the miRNAs may therefore be candidate novel biomarkers and therapeutic targets for a variety of diseases2,3,4,5. Although miRNA expression profiles in mouse organs and tissues including brain, heart, lung, liver, and kidney have been described6,7,8,9,10, there have been no standard methods for the extraction and evaluation of miRNAs in mouse kidney with renal interstitial fibrosis.
We have designed a protocol to reliably purify and detect the expressions of miRNAs in the kidneys of mice with renal interstitial fibrosis. The protocol involves five main steps, as follows. (1) 8-week-old C57BL/6 male mice are divided into groups of mice that undergo a sham-operation (controls) and mice that are subjected to a surgery providing unilateral ureteral obstruction (UUO), which is linked to renal interstitial fibrosis. (2) Kidney samples are extracted from the sham and UUO mice, homogenized separately in a silicon homogenizer, and then transferred to a biopolymer-shredding system on a microcentrifuge spin column11,12. (3) The total RNA containing miRNA is extracted from the kidney samples by a silica membrane-based spin column12,13. (4) Using this extracted total RNA, complementary DNA (cDNA) is synthesized from the total RNA with the use of reverse transcriptase, poly(A) polymerase, and oligo-dT primer14,15. (5) The expressions of miRNAs are evaluated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) using an intercalating dye14,15.
This protocol is based on investigations that obtained meaningful extractions and evaluations of miRNAs in a variety of tissues11,12,13,14,15, and the biopolymer-shredding system used in the protocol was shown to purify high-quality, total RNA from tissues in 200612. In addition, prior studies have confirmed the accuracy and sensitivity of aspects of the protocol (i.e., the cDNA synthesis with reverse transcriptase, poly(A) polymerase, and oligo-dT primers from extracted total RNA) for the determination of miRNA expression by qRT-PCR with an intercalating dye14,15. Since the new protocol has the advantages of simplicity, time-saving, and the reduction of technical errors, the protocol can be used in research that requires the accurate and sensitive identification of the miRNA profile in mouse kidney. Moreover, the protocol could be applied to investigations of many pathological conditions.
We next describe the determination of the miRNA expression profiles in mice with UUO, which is linked to renal interstitial fibrosis. In humans, renal interstitial fibrosis is a common and important feature of both chronic kidney disease and end-stage renal disease, regardless of their etiology16,17. This renal interstitial fibrosis is associated with the progression of renal failure, and it is characterized by increased expressions of extracellular matrix components in the interstitial spaces (e.g., collagen, fibronectin, and &#945;-smooth muscle actin)17,18.

<table><tbody><tr><td>Qiagen</td><td>79216</td><td>Wash buffer 2</td><td></td></tr><tr><td>Qiagen</td><td>1067933</td><td>Wash buffer 1</td><td></td></tr><tr><td>Tokyo Laboratory Animals Science</td><td>Not assigned</td><td></td><td></td></tr><tr><td>Thermo Fisher Scientific</td><td>4316813</td><td>96-well reaction plate</td><td></td></tr><tr><td>Thermo Fisher Scientific</td><td>4311971</td><td>Adhesive film for 96-well reaction plate</td><td></td></tr><tr><td>Qiagen</td><td>MS00001701</td><td>5&#039;-UUAAUGCUAAUUGUGAUAGGGGU-3&#039;</td><td></td></tr><tr><td>Qiagen</td><td>MS00065141</td><td>5&#039;-UUACACUCCAGUGGUGUCGGGU-3&#039;</td><td></td></tr><tr><td>Qiagen</td><td>MS00068067</td><td>5&#039;-UGCAGGGUUUAGUGUAGAGGG-3&#039;</td><td></td></tr><tr><td>Qiagen</td><td>MS00068081</td><td>5&#039;-UGUGUUAGAGCUCAGGGUUGAGA-3&#039;</td><td></td></tr><tr><td>Qiagen</td><td>217004</td><td>Membrane anchored spin column in a 2.0 mL collection tube</td><td></td></tr><tr><td>Qiagen</td><td>218161</td><td>Reverse transcriptase kit</td><td></td></tr><tr><td>Qiagen</td><td>218073</td><td>Green dye-based PCR kit</td><td></td></tr><tr><td>Qiagen</td><td>79654</td><td>Biopolymer spin columns in a 2.0 mL collection tube</td><td></td></tr><tr><td>Qiagen</td><td>79306</td><td>Phenol/guanidine-based lysis reagent</td><td></td></tr><tr><td>Thermo Fisher Scientific</td><td>4472380</td><td>Real-time PCR instrument</td><td></td></tr><tr><td>Thermo Fisher Scientific</td><td>4472380</td><td>Real-time PCR instrument software</td><td></td></tr><tr><td>Qiagen</td><td>129112</td><td></td><td></td></tr><tr><td>Qiagen</td><td>MS00033740</td><td>Not disclosed</td><td></td></tr><tr><td>Takara Bio</td><td>9790B</td><td>Silicon homogenizer</td><td></td></tr><tr><td>ASKUL</td><td>GA04SW</td><td></td><td></td></tr><tr><td>AS ONE</td><td>ER1004NA45-KF2,62 -9968-32</td><td></td><td></td></tr></tbody></table>

quantitative real-time pcr evaluation of microrna expressions in mouse kidney with unilateral ureteral obstruction

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3&#39; untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA&#39;s translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

A UUO mouse model was created by left ureteral ligation as described21 in 8-week-old male mice weighing 20&#8211;25 g. Ureters were completely obstructed by double ligation with 4-0 silk sutures. An analgesic (meloxicam 5 mg/kg, subcutaneous injection) was administered before surgery and also daily on the 2 days post-surgery. At 8 days post-surgery, kidneys were collected, rinsed with PBS, dissected, and stored in liquid nitrogen for further analysis. Sham-operated mice served as controls. The double ligation is successful if the left kidney has hydronephrosis. Based on the miRNA qRT-PCR data obtained using this UUO model, the level of miRNA-3070-3p was significantly increased and the levels of miRNA-7218-5p and miRNA-7219-5p were considerably decreased in the kidneys of the UUO mice compared to the controls (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61383/61383fig01.jpg" /> 
Figure 1: Differentially expressed miRNAs in the kidneys of UUO mice.&#160;qRT-PCR analysis of miRNA-3070-3p, miRNA-6401, miRNA-7218-5p, and miRNA-7219-5p expression in sham mice (n=8) and UUO mice (n=8). Values are mean &#177; standard error (error bars). T-tests were used to investigate significant differences between groups. T-tests with a p-value &#60;0.05 were considered significant. miRNA: microRNA, n.s.: not significant, qRT-PCR: quantitative real-time reverse-transcription polymerase chain reaction, UUO: unilateral ureteral obstruction. *p&#60;0.05. Click here to view a larger version of this figure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61383/61383fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction at JoVE.com

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

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.

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to ...

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Nanyang Technological University

Research

JoVE Journal

Genetics

6.2K Views.  Jichi Medical University. Deze methode maakt het mogelijk om microRNA expressie in nieren van muizen te voorzien van een breed scala aan pathologische aandoeningen, zoals nierfibrose. Het belangrijkste voordeel van deze techniek is dat het microRNA expressie profilering met hoge nauwkeurigheid en gevoeligheid mogelijk maakt met behulp van een eenvoudig proces dat tijd bespaart en technische fouten voorkomt. Om de schijnoperatie uit te voeren, gebruik je chirurgische schaar en pincet om een incisie in de huid in de bui...

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

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

Quantitative Real-Time Polymerase Chain Reaction Evaluation of MicroRNA Expression in Kidney and Serum of Mice with Age-Dependent Renal Impairment

MicroRNAs (miRNAs) are small, noncoding RNAs consisting of 21-25 bases. They are not translated into proteins but rather work to impede the functioning of their target messenger RNAs (mRNAs) by destabilizing them and disrupting their translation. Although the miRNA expression profiles in various mouse organs and tissues have been investigated, there have been no standard methods for purifying and quantifying mouse kidney and serum miRNAs. We have established an effective and reliable method for extracting and evaluating the miRNA expression in the serum and kidney of mice with age-dependent renal impairment. 
The method uses quantitative reverse-transcription-polymerase chain reaction (qRT-PCR), and the protocol requires six steps: (1) preparing senescence-accelerated mouse resistance 1 (SAMR1) mice and senescence-accelerated mouse prone (SAMP1) mice; (2) extracting serum samples from these mice; (3) extracting a kidney sample from each mouse; (4) extracting total RNA (including miRNA) from kidney and serum samples from each mouse; (5) the synthesis of complementary DNA (cDNA) with reverse transcription from the miRNA; (6) conducting a qRT-PCR using the cDNA obtained. 
This protocol was used to confirm that, compared to the controls, the expression of miRNA-7218-5p and miRNA-7219-5p was significantly changed in the kidney and serum of a mouse model of age-dependent renal impairment. This protocol also clarified the relationship between the kidney and serum of the mouse model of age-dependent renal impairment. This protocol can be used to determine miRNA expression in the kidney and serum of mice with age-dependent renal impairment.

We present a method for evaluating microRNA expression in the kidney and serum of mice with age-dependent renal impairment by quantitative reverse-transcription polymerase chain reaction.

MicroRNAs (miRNAs) are small, noncoding RNAs consisting of 21-25 bases. They are not translated into proteins but rather work to impede the functioning of ...

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

Medicine

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

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal failure. In recent years, the involvement of microRNAs (miRNAs) has been reported in the pathogenesis of IgA nephropathy. However, there is no established method for profiling miRNAs in IgA nephropathy using small animal models. Therefore, we developed a reliable method for analyzing miRNA in the kidney of an IgA mouse model (HIGA mouse). The goal of this protocol is to detect the altered expression levels of miRNAs in the kidneys of HIGA mice when compared with the levels in kidneys of control mice. In brief, this method consists of four steps: 1) obtaining kidney samples from HIGA mice; 2) purifying total RNA from kidney samples; 3) synthesizing complementary DNA from total RNA; and 4) quantitative reverse transcription polymerase chain reaction (qRT-PCR) of miRNAs. Using this method, we successfully detected the expression levels of several miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p) in the kidneys of HIGA mice. This new method can be applied to other studies profiling miRNAs in IgA nephropathy.

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.

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal ...

Immunology and Infection

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.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

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 ...

Biology

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

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, standard protocols for the purification of miRNAs and detection of their expression in the kidneys of acute kidney injury (AKI) mice have not been well established. This study developed an effective and simple protocol to purify and quantify miRNAs in the kidneys of an AKI mouse model induced by renal ischemia-reperfusion using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). This protocol comprises five steps: 1) induction of AKI by renal ischemia-reperfusion, 2) harvesting of kidneys, 3) purification of total RNA, including miRNAs, from kidneys, 4) cDNA synthesis by reverse transcription of miRNA, and 5) qRT-PCR to detect miRNA expression. Using this protocol, the renal ischemia-reperfusion injury model can be generated with mild to severe forms of AKI. Additionally, if the procedure is followed properly, a consistent AKI model with minimal individual differences can be obtained. This qRT-PCR assay shows a very wide dynamic range and enables the discrimination of mature miRNAs, which can be accurately quantified with high specificity. This protocol can be used to study the miRNA expression profile in AKI kidneys.

This article presents a protocol for detecting microRNA expression in the kidneys of an acute kidney injury mouse model using quantitative real-time reverse-transcription polymerase chain reaction. This protocol emphasizes an ischemic kidney injury mouse model and the careful extraction of microRNA samples.

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

An Immature Murine Model of Reversible Unilateral Ureteral Obstruction

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ultimately resulting in irreversible kidney damage. The alleviation of UUO is crucial. Several animal models of reversible unilateral ureteral obstruction (RUUO) have been established in the literature, enabling the observation of structural changes and functional damage while also simulating physiological and pathophysiological changes following the relief of ureteral obstruction. In this study, a reversible obstruction model was established in the unilateral murine ureter using a silicone tube. Significant renal damage was observed prior to obstruction relief, with partial recovery noted afterward. Unlike UUO, this model prevents progressive hydronephrosis, leading to distinct pathological outcomes. This simple surgical procedure demonstrates a high success rate and holds promise as a classical model for investigating reversible obstructive nephropathy and potential treatments for renal interstitial fibrosis. Furthermore, it provides a practical platform for studying the mechanisms of recovery from obstructive nephropathy, renal cell regeneration, and tissue remodeling.

The present protocol describes a step-by-step, reproducible model of unilateral ureteral obstruction.

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ...

Unilateral Ureteral Obstruction Model for Investigating Kidney Interstitial Fibrosis

Kidney fibrosis is the final pathological outcome of progressive chronic kidney disease (CKD). The unilateral ureteral obstruction (UUO) model is widely used to elucidate the molecular and cellular mechanisms underlying kidney interstitial fibrosis and to identify potential therapeutic targets. This model is established in mice through surgical ligation of a unilateral ureter, a procedure that is relatively straightforward and easy to perform. However, the UUO mouse model is known to exhibit significant variability and inconsistency, influenced by factors such as mouse strain, age, sex, anesthesia type, duration of surgery, body temperature during the procedure, the operator's surgical skills, feeding conditions, and the overall health status of the mice. Variations in surgical techniques, suture placement, and the duration of obstruction contribute to the variability in outcomes. Additionally, inconsistent sampling of obstructed kidneys further increases variability in the assessment of kidney fibrosis. This study outlines the process of developing the UUO mouse model and evaluating interstitial fibrosis, discusses the technical challenges contributing to the model's unpredictability, and proposes potential solutions. These insights aim to establish a more standardized and universally applicable approach for investigating kidney fibrosis.

This protocol describes a method for inducing unilateral ureteral obstruction (UUO) in mice to study the progression of tissue fibrosis in obstructive nephropathy. It includes surgical procedures, post-operative care, and methods for fibrosis assessment.

Kidney fibrosis is the final pathological outcome of progressive chronic kidney disease (CKD). The unilateral ureteral obstruction (UUO) model is widely ...

Unilateral Ureteral Obstruction Murine Model: A Surgical Procedure to Obstruct the Urinary Flow from a Kidney in Mouse Models

Source: Yanai, K. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/61383" target="_blank">Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction.</a> J. Vis. Exp. (2020)
This video demonstrates the procedure to generate a unilateral ureteral obstruction mouse model. The procedure involves cutting one of the ureters of the mouse to obstruct urine flow.

Source: Yanai, K. et al. Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction. J. Vis. Exp. ...

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

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