We thank Sarah Williams, PhD, from Edanz Group (www.edanzediting.com) for editing a draft of this manuscript.

Animal experiments were approved by the Animal Ethics Committee of Jichi Medical University and comply with the Use and Care of Experimental Animals guidelines from the Jichi Medical University Guide for Laboratory Animals.
1. Obtaining kidney samples from HIGA mice
NOTE: HIGA mice show a stable phenotype of IgA nephropathy after 25 weeks of age8,9,10,11. Balb/c mice should be selected as the control group8,9,10,11. 25-week-old female HIGA mice (n = 10) and 25-week-old female Balb/c mice (n = 10) were obtained. It is necessary to determine the number of mice required for experiments in advance. This step requires about 7&#8211;8 h for a sample size of 10 HIGA mice and 10 Balb/c mice.
<ol>
	<li>Prepare these items: HIGA mice (25-week-old, female), Balb/c mice (25-week-old, female), inhalation anesthesia apparatus, isoflurane, cork sheet, pin, phosphate-buffered saline (PBS), injection syringes, needles, surgical scissors, and forceps.</li>
	<li>Anesthetize a HIGA mouse using 4%&#8211;5% of isoflurane with inhalation anesthesia apparatus and mount it in the dorsal position on the cork sheet using pins. Adjust the concentration of isoflurane to 2%&#8211;3% as the maintenance dose after the induction of anesthesia. 
	NOTE: The depth of anesthesia is maintained at a level at which the pain associated with the invasive procedure is completely eliminated. In particular, isoflurane concentration should be adjusted to 4-5% when excising tissues such as the thorax. Hair removal and eye ointment are not essential. It is not necessary to mount the mouse on a heating pad if the procedure can be performed quickly.</li>
	<li>Make a 3&#8211;4 cm midline incision of the mouse abdominal wall with surgical scissors and forceps and carefully identify both sides of the kidney.</li>
	<li>Incise the ribs and diaphragm with surgical scissors and forceps to expose the heart. After incising the right atrium, inject PBS into the left ventricle until the kidney color changes to pale yellow (this procedure means that the whole body of the mouse is perfused with PBS.)</li>
	<li>Cut the renal artery, renal vein, and ureter, and remove the kidney. Divide the kidney into 30 mg pieces for use in the next step. 
	NOTE: The pathology of IgA nephropathy mainly involves the glomerulus in the renal cortex (outer part of the kidney). Although it is difficult to macroscopically and completely distinguish the cortex and the medulla, it is desirable to mainly collect the outer part of the kidney. These samples can be stored at &#8211;80 &#176;C for several months.</li>
</ol>
2. Purifying total RNA from kidney samples
NOTE: In this step, commercially available miRNA isolation kit is used for the extraction of total RNA. In addition, biopolymer-shredding spin column is used. See Table of Materials for additional details. miRNA isolation kit contains a silica membrane-based spin column, phenol/guanidine-based lysis reagents, guanidine/ethanol wash buffer (wash buffer 1), ethanol wash buffer (wash buffer 2), and nuclease-free water. This step requires about 3 h for a sample size of 10 HIGA mice and 10 control mice.
<ol>
	<li>Prepare the following items: 1.5 or 2.0 mL collection tubes, 100% ethanol, chloroform, silicon homogenizer, micropipettes, pipette tips, centrifuge, biopolymer-shredding spin column, silica membrane-based spin column, phenol/guanidine-based lysis reagents, guanidine/ethanol wash buffer (wash buffer 1), ethanol wash buffer (wash buffer 2), and nuclease-free water.</li>
	<li>Homogenize 30 mg kidney samples using a silicon homogenizer and 700 &#181;L of the phenol/guanidine-based lysis reagent at room temperature. 
	NOTE: The miRNAs in the sample are vulnerable until they have been exposed to phenol/guanidine-based lysis reagent so these steps should be performed promptly.</li>
	<li>Transfer the lysate to the biopolymer-shredding spin column and centrifuge it at 15,000 x g for 2 min at room temperature.</li>
	<li>Add 140 &#181;L of chloroform to the filtrate and mix vigorously for 15 s. Leave for 2&#8211;3 min, then centrifuge at 12,000 x g at 4 &#176;C for 15 min.</li>
	<li>Gently transfer the clear supernatant without touching the middle layer to a new collection tube and add the determined volume (see below) of 100% ethanol. Vortex for 5 s. 
	NOTE: 100% ethanol at 1.5x the volume of the obtained supernatant is required.</li>
	<li>Transfer the sample (upper limit 700 &#181;L) to a silica membrane-based spin column and centrifuge the sample at room temperature for 15 s at 8,000 x g. Discard the filtrate after centrifugation.</li>
	<li>Add 700 &#181;L of wash buffer 1 to the silica membrane-based spin column and centrifuge the column at room temperature for 15 s at 8,000 x g. Discard the filtrate after centrifugation.</li>
	<li>Add 500 &#181;L of wash buffer 2 to the silica membrane-based spin column and centrifuge it at room temperature for 15 s at 8,000 x g. Discard the filtrate after centrifugation.</li>
	<li>Add 500 &#181;L of wash buffer 2 to the silica membrane-based spin column and centrifuge it at room temperature for 15 s at 8,000 x g. Discard the filtrate after centrifugation.</li>
	<li>Centrifuge the silica membrane-based spin column again without adding anything to remove any additional ethanol, at 15,000 x g for 1 min at room temperature. Throw away the filtrate after centrifugation.</li>
	<li>Change the tube attached to the spin column to a new collection tube.</li>
	<li>Add 30 &#181;L of nuclease-free water to the silica membrane-based spin column and centrifuge it at 8,000 x g for 1 min at room temperature. 
	NOTE: The resulting 30 &#181;L solution contains a high concentration of total RNA. This sample can be stored at &#8211;80 &#176;C for several months.</li>
</ol>
3. Synthesis of cDNA from total RNA
NOTE: In this step, a commercially available reverse transcription kit is used. See Table of Materials for additional details. This kit contains nucleic acid mix, reverse transcriptase mix, and buffer. This procedure must be performed on ice to prevent progress of the reaction. This step requires about 3 h for a sample size of 10 HIGA mice and 10 control mice.
<ol>
	<li>Prepare the following items: 1.5 mL collection tubes, eight-well strip tubes, micropipettes, pipette tips, spectrophotometer, nuclease-free water, ice, nucleic acid mix, reverse transcriptase mix, buffer, and thermal cycler.</li>
	<li>Measure the concentration of total RNA using a spectrophotometer. Then, calculate the required volume of nuclease-free water so that 12 &#181;L contains 1 &#181;g of total RNA.</li>
	<li>Prepare a master mix with the following contents: 2.0 &#181;L of nucleic acid mix, 2.0 &#181;L of reverse transcriptase mix, and 4.0 &#181;L of buffer to a total of 8.0 &#181;L per sample.</li>
	<li>Add 8 &#181;L of the master mix to each well of eight-well strip tubes.</li>
	<li>Dilute total RNA in the volume of nuclease-free water calculated in 3.2. Then, add 12 &#181;L of the total RNA solution (containing 1 &#181;g of total RNA) to each well of eight-well strip tubes.</li>
	<li>Incubate the sample in 8 well strip tubes at 37 &#176;C for 60 min using the thermal cycler. Then, incubate at 95 &#176;C for 5 min using the thermal cycler. 
	NOTE: The solution after incubation contains a high concentration of cDNA.</li>
	<li>Transfer this solution to a 1.5 mL tube and add 200 &#181;L of nuclease-free water. 
	NOTE: The resulting 200 &#181;L of solution can be used for qRT-PCR as a template cDNA. This sample can be stored at &#8211;80 &#176;C for about 1 year.</li>
</ol>
4. qRT-PCR of miRNA
NOTE: In this step, a commercially available PCR kit is used. See Table of Materials for additional details. This kit contains PCR mix, universal primer, and nuclease-free water. Samples should be prepared in duplicate, and the accuracy of the results should be considered in each case. Expression levels of miRNA are quantified by the &#916;&#916;CT method. This step requires about 4 h for a sample size of 10 HIGA mice and 10 control mice.
<ol>
	<li>Prepare the following items: 1.5 mL collection tube, 96-well reaction plate, adhesive film for the 96-well reaction plate, micropipettes, pipette tips, PCR mix, universal primer, nuclease-free water, miRNA-specific primers, and a real-time PCR instrument.</li>
	<li>Prepare the master mix with the following contents: 12.5 &#181;L of PCR mix, 2.5 &#181;L of universal primer, 1.25 &#181;L of 5 &#181;M miRNA-specific primer, and 6.25 &#181;L of nuclease-free water for each well. 
	NOTE: In this assay, primers specific to miRNAs [RNA, U6 Small Nuclear 2 (RNU6-2), miR-155-5p, miR-146a-5p, and miR-21-5p] were used. RNU6-2 was used as an endogenous control14.</li>
	<li>Add 22.5 &#181;L of the master mix to each well of the 96 well reaction plate.</li>
	<li>Add 2.5 &#181;L of cDNA prepared in step 3.7 to individual well of the 96 well plate.</li>
	<li>Cover the 96 well reaction plate with adhesion film, then centrifuge at 1,000 x g for 30 s.</li>
	<li>Run the real-time PCR instrument. Program the PCR instrument as follows: initial activation at 95 &#176;C for 15 min, then 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>Quantify gene expression using the &#916;&#916;CT method. Relative expression levels are determined as 2&#8211;&#916;&#916;CT. 
	NOTE: Abnormal PCR amplification curves should be considered for exclusion from the results.</li>
</ol>

<ol>
	<li>Rodrigues, J. C., Haas, M., Reich, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgA+Nephropathy.">IgA Nephropathy.</a> Clinical Journal of the American Society of Nephrology. 12 (4), 677-686 (2017).</li><li>Szeto, C. C., Li, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+IgA+nephropathy.">MicroRNAs in IgA nephropathy.</a> Nature Reviews Nephrology. 10 (5), 249-256 (2014).</li><li>Wyatt, R. J., Julian, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgA+nephropathy.">IgA nephropathy.</a> The New England Journal of Medicine. 368 (25), 2402-2414 (2013).</li><li>Vishnoi, A., Rani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRNA+Biogenesis+and+Regulation+of+Diseases:+An+Overview.">MiRNA Biogenesis and Regulation of Diseases: An Overview.</a> Methods in Molecular Biology. 1509, 1-10 (2017).</li><li>Rupaimoole, R., Slack, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+therapeutics:+towards+a+new+era+for+the+management+of+cancer+and+other+diseases.">MicroRNA therapeutics: towards a new era for the management of cancer and other diseases.</a> Nature Reviews Drug Discovery. 16 (3), 203-222 (2017).</li><li>Serino, G., Sallustio, F., Cox, S. N., Pesce, F., Schena, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+miR-148b+expression+promotes+aberrant+glycosylation+of+IgA1+in+IgA+nephropathy.">Abnormal miR-148b expression promotes aberrant glycosylation of IgA1 in IgA nephropathy.</a> Journal of the American Society of Nephrology. 23 (5), 814-824 (2012).</li><li>Serino, G., 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=In+a+retrospective+international+study,+circulating+miR-148b+and+let-7b+were+found+to+be+serum+markers+for+detecting+primary+IgA+nephropathy.">In a retrospective international study, circulating miR-148b and let-7b were found to be serum markers for detecting primary IgA nephropathy.</a> Kidney International. 89 (3), 683-692 (2016).</li><li>Muso, E., 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=Enhanced+production+of+glomerular+extracellular+matrix+in+a+new+mouse+strain+of+high+serum+IgA+ddY+mice.">Enhanced production of glomerular extracellular matrix in a new mouse strain of high serum IgA ddY mice.</a> Kidney International. 50 (6), 1946-1957 (1996).</li><li>Kurano, M., Yatomi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+gas+chromatography+mass+spectrometry+to+elucidate+metabolites+predicting+the+phenotypes+of+IgA+nephropathy+in+hyper+IgA+mice.">Use of gas chromatography mass spectrometry to elucidate metabolites predicting the phenotypes of IgA nephropathy in hyper IgA mice.</a> Plos One. 14 (7), 0219403 (2019).</li><li>Hyun, Y. Y., 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=Adipose-derived+stem+cells+improve+renal+function+in+a+mouse+model+of+IgA+nephropathy.">Adipose-derived stem cells improve renal function in a mouse model of IgA nephropathy.</a> Cell Transplantation. 21 (11), 2425-2439 (2012).</li><li>Katsuma, S., 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=Genomic+analysis+of+a+mouse+model+of+immunoglobulin+A+nephropathy+reveals+an+enhanced+PDGF-EDG5+cascade.">Genomic analysis of a mouse model of immunoglobulin A nephropathy reveals an enhanced PDGF-EDG5 cascade.</a> The Pharmacogenomics Journal. 1 (3), 211-217 (2001).</li><li>Morse, S. M., Shaw, G., Larner, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+mRNA+and+protein+extraction+from+the+same+experimental+sample+using+a+commercially+available+column-based+RNA+preparation+kit.">Concurrent mRNA and protein extraction from the same experimental sample using a commercially available column-based RNA preparation kit.</a> BioTechniques. 40 (1), 54-58 (2006).</li><li>Mestdagh, P., 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=Evaluation+of+quantitative+miRNA+expression+platforms+in+the+microRNA+quality+control+(miRQC)+study.">Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study.</a> Nature Methods. 11 (8), 809-815 (2014).</li><li>Sauer, E., Babion, I., Madea, B., Courts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evidence+based+strategy+for+normalization+of+quantitative+PCR+data+from+miRNA+expression+analysis+in+forensic+organ+tissue+identification.">An evidence based strategy for normalization of quantitative PCR data from miRNA expression analysis in forensic organ tissue identification.</a> Forensic Science International: Genetics. 13, 217-223 (2014).</li><li>Wang, G., 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=Elevated+levels+of+miR-146a+and+miR-155+in+kidney+biopsy+and+urine+from+patients+with+IgA+nephropathy.">Elevated levels of miR-146a and miR-155 in kidney biopsy and urine from patients with IgA nephropathy.</a> Disease Markers. 30 (4), 171-179 (2011).</li><li>Hennino, M. F., 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=miR-21-5p+renal+expression+is+associated+with+fibrosis+and+renal+survival+in+patients+with+IgA+nephropathy.">miR-21-5p renal expression is associated with fibrosis and renal survival in patients with IgA nephropathy.</a> Scientific Reports. 6, 27209 (2016).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> How to Win the Battle with RNase. 2019 (2), (2019).</li></ol>

The authors declare that they have no conflicts of interest.

We were able to measure the expression levels of miRNAs in the kidneys of an IgA nephropathy mouse model (HIGA mice) using this new method. IgA nephropathy is an unexplained disease that needs further research to clarify its etiology and therapeutic targets1,3. However, obtaining human kidney samples is highly invasive. This new technique is advantageous in that it allows the study of IgA nephropathy using small animals and should facilitate future research into IgA nephropathy and miRNAs. In a future study, this method could be applied to the study of novel miRNAs for the treatment of IgA nephropathy. Artificial modulation of miRNAs in HIGA mice may influence the phenotype of IgA nephropathy. In that case, this method may be useful for verifying the changes of miRNAs. This method is not intended for analyzing miRNAs in serum, but it may be possible to use it for this by modifying the protocol.
The rationale of this method is based on previous reports. We used biopolymer-shredding spin columns to disrupt biopolymers in the sample, which has previously been shown to be an effective technique12. Past reports have also demonstrated the accuracy of synthesizing cDNA from total RNA using reverse transcription and performing PCR using the prepared cDNA as a template13. As a critical step in this method, evaluation of the PCR results is important. It is necessary to exclude abnormal amplification curves from the results. In addition, changes in endogenous controls should be considered if stable results are not obtained14.
A major problem that can be encountered with this method is the abnormally low expression of miRNAs. miRNAs are highly degradable compounds so the method should be performed quickly. Additionally, the effects of ribonuclease (RNase) in the experimental environment need to be eliminated. Researchers should always be aware of the presence of RNase in skin and hair17. It is necessary to wear disposable gloves, masks, and hair caps for experiments. In addition, laboratory equipment such as micropipettes, pipette tips, and collection tubes must be cleaned to ensure the absence of RNase17. The deactivation of RNase using an autoclave should be performed as necessary17. Otherwise, researchers should use RNase-free-certified items17. All samples should be properly stored under the recommended conditions to reduce the risk of RNase contamination.
Our method has three important limitations. The first is that we did not demonstrate whether it can be applied to other animals and other organs. This is relevant because miRNAs are known to play an important role in blood cells in IgA nephropathy2,6,7, but we did not investigate whether our method can be used in blood cells. Second, the sample size of our experiments may be small. Sample size should be determined depending on the study design, statistical analysis, required time, and required costs. Therefore, each researcher must determine the appropriate sample size before starting this experiment. Third, the severity and phenotype of IgA nephropathy in HIGA mice may vary between individuals9. In addition, the bias of sex and age has not been fully investigated. It is recommended to add immunohistochemistry to confirm the severity and phenotype of IgA nephropathy, if necessary. In addition, the methods other than PCR, including microarray, northern blotting, and ribonuclease protection assays, are recommended to perform as necessary.
In conclusion, we developed a reliable method for measuring the expression levels of miRNAs in the kidneys of an IgA mouse model (HIGA mice).

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA in the renal glomerular mesangial region1,2. It is the most common of the primary glomerulonephritis and leads to the end-stage renal failure in 20%&#8211;40% of patients2. The cause is still unknown but persistent mucosal infection has been implicated1,3. Corticosteroids, immunosuppressants, and renin&#8722;angiotensin system inhibitors have been proposed as therapeutic methods1,3, but have not been completely established3. Therefore, further research is required to clarify the etiology and therapeutic methods of treating IgA nephropathy.
microRNAs (miRNAs) are small, non-coding RNAs that play an important role in regulating gene expression4,5. miRNAs are reported to be involved in the pathogenesis of various diseases, and some have been identified as disease biomarkers and therapeutic agents4,5. In recent years, an association between miRNAs and the pathogenesis of IgA nephropathy has also been reported2,6,7. For example, miR-148b was shown to be involved in structural abnormalities of IgA in patients with IgA nephropathy2,6,7, while miR-148b and let-7b were documented as novel biomarkers for detecting IgA nephropathy7. Although understanding the effects of miRNAs on IgA nephropathy may help further elucidate etiology and treatment2, standard methods for profiling miRNAs in IgA nephropathy using small animal models have not yet been established2.
We herein developed a simple and reliable method for measuring miRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). The HIGA mouse is a characteristic ddY strain showing a particularly high level of serum IgA and the abnormal deposition of IgA in kidney glomeruli8,9,10,11. Therefore, HIGA mice can be used as an IgA nephropathy mouse model8,9,10,11. Our method consists of four major steps: first, surgically obtaining kidney samples from HIGA mice; second, homogenizing samples and purifying total RNA using a silica membrane-based spin column; third, synthesizing complementary DNA (cDNA) from total RNA using reverse transcription; and fourth, detecting the expression levels of miRNA by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The rationale for this method and the reliability of the results are based on previous reports12,13. We show that this is a useful technique to accurately measure miRNA expression levels in an IgA nephropathy mouse model, and that it could be used to facilitate future research into miRNAs in IgA nephropathy.

<table>
	<tbody>
		<tr>
			<td>BALB/cCrSlc (25-week-old, female)</td>
			<td>Japan SLC, Inc.</td>
			<td>none</td>
			<td>Mouse for control</td>
		</tr>
		<tr>
			<td>HIGA/NscSlc (25-week-old, female)</td>
			<td>Japan SLC, Inc.</td>
			<td>none</td>
			<td>IgA nephropathy mouse model</td>
		</tr>
		<tr>
			<td>MicroAmp Optical 96 well reaction plate for qRT-PCR</td>
			<td>Thermo Fisher Scientific</td>
			<td>4316813</td>
			<td>96-well reaction plate</td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive Film</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311971</td>
			<td>adhesive film for 96-well reaction plate</td>
		</tr>
		<tr>
			<td>miScript II RT kit</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td>Experimental kit for synthesis of cDNA</td>
		</tr>
		<tr>
			<td>miRNeasy Mini kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td>Experimental kit fot extraction of total RNA</td>
		</tr>
		<tr>
			<td>miScript Primer Assay (RNU6-2)</td>
			<td>Qiagen</td>
			<td>MS00033740</td>
			<td>miRNA-specific primer</td>
		</tr>
		<tr>
			<td>miScript Primer Assay (miR-155-5p)</td>
			<td>Qiagen</td>
			<td>MS00001701</td>
			<td>miRNA-specific primer</td>
		</tr>
		<tr>
			<td>miScript Primer Assay (miR-146a-5p)</td>
			<td>Qiagen</td>
			<td>MS00001638</td>
			<td>miRNA-specific primer</td>
		</tr>
		<tr>
			<td>miScript Primer Assay (miR-21-5p)</td>
			<td>Qiagen</td>
			<td>MS00009079</td>
			<td>miRNA-specific primer</td>
		</tr>
		<tr>
			<td>miScript SYBR Green PCR kit</td>
			<td>Qiagen</td>
			<td>218073</td>
			<td>Experimental kit for real-time PCR</td>
		</tr>
		<tr>
			<td>QIA shredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>biopolymer-shredding spin column</td>
		</tr>
		<tr>
			<td>QuantStudio 12K Flex Flex Real-Time PCR system</td>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>real-time PCR instrument</td>
		</tr>
		<tr>
			<td>QuantStudio 12K Flex Software version 1.2.1.</td>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>real-time PCR instrument software</td>
		</tr>
		<tr>
			<td>takara biomasher standard</td>
			<td>Takara Bio</td>
			<td>9790B</td>
			<td>silicon homogenizer</td>
		</tr>
	</tbody>
</table>

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.

We investigated the expression levels of miRNAs in the kidneys of HIGA mice (n=10). This result was obtained completely based on the described protocol. The kidneys of Balb/c mice were selected as the control (n=10). In both groups, aged 25 weeks were selected. Only female HIGA mice were available from the supplier. The expression levels of three miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p; Figure 1) were detected, which were previously reported to be associated with IgA nephropathy15,16. Relative miRNA expression levels of the two groups were compared using a t-test, with P &#60; 0.05 being statistically significant. miR-155-5p was expressed at 3.3-fold higher levels in the HIGA group compared with the control group (P &#60; 0.001), while miR-21-5p was expressed at 1.58-fold higher levels in the HIGA group (P = 0.007). miR-146a-5p expression did not differ significantly between the two groups.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61535/61535fig01.jpg" /> 
Figure 1: Expression levels of miRNAs in the kidneys of HIGA mice.&#160;qRT-PCR determined the relative expression levels of miR-155-5p, miR-146a-5p, and miR-21-5p in HIGA mice (n = 10) and Balb/c mice (n = 10). Relative expression levels are shown as mean values and standard errors. (A) miR-155-5p expression was 3.3-fold higher in the HIGA group (P &#60; 0.001). (B) miR-146a-5p expression did not differ significantly between the two groups. (C) miR-21-5p expression was 1.58-fold higher in the HIGA group (P = 0.007). quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR); microRNAs (miRNAs); not significant (NS); *, p &#60;0.05. <a href="https://www.jove.com/files/ftp_upload/61535/61535fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice at JoVE.com

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

1.8K Views.  Jichi Medical University. 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.

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

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.