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,<sup class="x...

<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. 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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>

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 ...

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 nephropathy<sup c...

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 ...

This method can be used to easily verify the change of microRNAs in IGA nephropathy using a small animal model, making it possible to study IGA nephropathy without a human kidney biopsy. The protocol can provide new insights into the genetics of IGA nephropathy, which can potentially aid in discovery of new diagnostic and new therapeutic methods for the kidney disease. After ensuring that the mouse is properly anesthetized, use surgical scissors and forceps to make a three-to four-centimeter midline incision in the abdominal wall of the mouse and identify both sides of the kidney.Incise the ribs and diaphragm to expose the heart. After incising the right atrium, inject PBS into the left ventricle until the kidney color changes to pale yellow, indicating that the whole body of the mouse is perfused with PBS. Cut the renal artery, renal vein, and ureter, then remove the kidney.Homogenize the 30-milligram kidney samples using a silicon homogenizer and 700 microliters of phenol guanidine-based lysis reagent at room temperature. Transfer the lysate to the biopolymer shredding spin column and centrifuge it at 15, 000 G for two minutes at room temperature. Continue with RBNA purification following manuscript directions.After the final wash, add 30 microliters of nuclease-free water to the silica membrane-based spin column and centrifuge it at 8, 000 G for one minute to elute the RNA. For cDNA synthesis, prepare a master mix with two microliters of nucleic acid mix, two microliters of reverse transcriptase mix, and four microliters of buffer in a total of eight microliters per sample. Add eight microliters of the master mix to each well of an eight-well tube strip.Dilute total RNA in nuclease-free water. Then, add 12 microliters of the total RNA solution to each well of the strip. Place the samples in the thermocycler and incubate them at 37 degrees Celsius for 60 minutes, then at 95 degrees Celsius for five minutes.To perform quantitative real-time PCR, prepare the master mix according to manuscript directions, then add 22.5 microliters of the master mix to each well of a 96-well reaction plate. Add 2.5 microliters of the prepared cDNA to each well. Place the reaction plate in the real-time PCR instrument.Then, program it for an initial activation at 95 degrees Celsius for 15 minutes, followed by 40 cycles of denaturation at 94 degrees Celsius for 15 seconds, annealing at 55 degrees Celsius for 30 seconds, and extension at 70 degrees Celsius for 30 seconds. Quantify gene expression using the delta-delta CT method. This protocol was used to investigate the expression levels of microRNAs in the kidneys of HIGA mice using the kidneys of Balb/c mice as control.The expression levels of three IGA nephropathy-associated microRNAs were measured. Relative microRNA expression levels of the two groups were compared using a T-test with a P of less than 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, and miR-21-5p was expressed at 1.58-fold higher levels.miR-1461-5p expression did not differ significantly between the two groups. When attempting this protocol, it is important to wear a mask, gloves, and cap and to keep the laboratory table and tools clean in order to prevent nuclease in the environment from affecting the experiment. This experimental method is currently in practical use in our laboratory.It's goal, to establish new diagnostic and therapeutic methods for IGA nephropathy.

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Immunology and Infection

1.7K vues.  Jichi Medical University. This method can be used to easily verify the change of microRNAs in IGA nephropathy using a small animal model, making it possible to study IGA nephropathy without a human kidney biopsy. The protocol can provide new insights into the genetics of IGA nephropathy, which can potentially aid in discovery of new diagnostic and new therapeutic methods for the kidney disease. After ensuring that the mouse is properly anesthetized, use surgical scissors and forceps to make a three-to four-centimeter ...