Name: detection of microrna expression in the kidneys of immunoglobulin a nephropathic mice
Uploaded: 2020-07-08T14:00:00
Description: Watch this Scientific Journal Video about Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice at JoVE.com

Vi takker Sarah Williams, ph.d., fra Edanz Group (www.edanzediting.com) for at redigere et udkast til dette manuskript.

Dyreforsøg blev godkendt af Animal Ethics Committee of Jichi Medical University og overholder retningslinjerne for brug og pleje af forsøgsdyr fra Jichi Medical University Guide for Laboratory Animals.
1. Indhentning af nyreprøver fra HIGA-mus
BEMÆRK: HIGA mus viser en stabil fænotype af IgA nefropati efter 25 ugers alderen 8,9,10,11. B...

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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>

Vi var i stand til at måle ekspressionsniveauerne af miRNA'er i nyrerne hos en IgA nefropati musemodel (HIGA mus) ved hjælp af denne nye metode. IgA nefropati er en uforklarlig sygdom, der kræver yderligere forskning for at afklare dens ætiologi og terapeutiske mål 1,3. Imidlertid er opnåelse af humane nyreprøver meget invasiv. Denne nye teknik er fordelagtig, fordi den tillader undersøgelse af IgA-nefropati ved hjælp af små dyr og bør lette fremtidig ...

Immunoglobulin A (IgA) nefropati er en type primær glomerulonefritis karakteriseret ved den unormale aflejring af IgA i den renale glomerulære mesangialregion 1,2. Det er den mest almindelige af den primære glomerulonefritis og fører til nyresvigt i slutstadiet hos 20%-40% af patienterne2. Årsagen er stadig ukendt, men vedvarende slimhindeinfektion er impliceret 1,3. Kortikosteroider, immunsuppressiva og renin-angiotensinsystemhæmmere er blevet foreslået som terapeutiske metoder1,3, men er ikke blevet fuldstændigt etableret3. Derfor er der behov for yderligere forskning for at afklare ætiologien og terapeutiske metoder til behandling af IgA nefropati.
microRNA'er (miRNA'er) er små, ikke-kodende RNA'er, der spiller en vigtig rolle i reguleringen af genekspression 4,5. miRNA'er rapporteres at være involveret i patogenesen af forskellige sygdomme, og nogle er blevet identificeret som sygdomsbiomarkører og terapeutiske midler 4,5. I de senere år er der også rapporteret en sammenhæng mellem miRNA'er og patogenesen af IgA-nefropati 2,6,7. For eksempel viste miR-148b sig at være involveret i strukturelle abnormiteter af IgA hos patienter med IgA nefropati 2,6,7, mens miR-148b og let-7b blev dokumenteret som nye biomarkører til påvisning af IgA nefropati7. Selvom forståelse af virkningerne af miRNA'er på IgA-nefropati kan hjælpe yderligere med at belyse ætiologi og behandling 2, er standardmetoder til profilering af miRNA'er i IgA-nefropati ved hjælp af små dyremodeller endnu ikke etableret2.
Vi har heri udviklet en enkel og pålidelig metode til måling af miRNA ekspressionsniveauer i nyrerne hos en IgA nefropati musemodel (HIGA mus). HIGA musen er en karakteristisk ddY-stamme, der viser et særligt højt niveau af serum IgA og den unormale aflejring af IgA i nyreglomeruli 8,9,10,11. Derfor kan HIGA-mus bruges som en IgA nefropati musemodel 8,9,10,11. Vores metode består af fire hovedtrin: For det første kirurgisk opnåelse af nyreprøver fra HIGA-mus; for det andet homogenisering af prøver og rensning af total RNA ved anvendelse af en silicamembranbaseret spinsøjle; for det tredje syntetisering af komplementært DNA (cDNA) fra total RNA ved anvendelse af revers transkription; og for det fjerde detektering af ekspressionsniveauerne af miRNA ved kvantitativ omvendt transkriptionspolymerasekædereaktion (qRT-PCR). Begrundelsen for denne metode og resultaternes pålidelighed er baseret på tidligere rapporter12,13. Vi viser, at dette er en nyttig teknik til nøjagtigt at måle miRNA-ekspressionsniveauer i en IgA-nefropati-musemodel, og at den kan bruges til at lette fremtidig forskning i miRNA'er i IgA-nefropati.

<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) nefropati er en type primær glomerulonefritis karakteriseret ved den unormale aflejring af IgA, hvilket fører til nyresvigt i slutstadiet. I de senere år er involveringen af mikroRNA'er (miRNA'er) blevet rapporteret i patogenesen af IgA-nefropati. Der er imidlertid ingen etableret metode til profilering af miRNA'er i IgA-nefropati ved hjælp af små dyremodeller. Derfor udviklede vi en pålidelig metode til analyse af miRNA i nyren af en IgA-musemodel (HIGA-mus). Målet med denne protokol er at detektere de ændrede ekspressionsniveauer af miRNA'er i nyrerne hos HIGA-mus sammenlignet med niveauerne i nyrerne hos kontrolmus. Kort fortalt består denne metode af fire trin: 1) opnåelse af nyreprøver fra HIGA-mus; 2) oprensning af total RNA fra nyreprøver; 3) syntetisering af komplementært DNA fra total RNA; og 4) kvantitativ revers transkriptionspolymerasekædereaktion (qRT-PCR) af miRNA'er. Ved hjælp af denne metode detekterede vi med succes ekspressionsniveauerne for flere miRNA'er (miR-155-5p, miR-146a-5p og miR-21-5p) i nyrerne hos HIGA-mus. Denne nye metode kan anvendes til andre undersøgelser, der profilerer miRNA'er i IgA-nefropati.

Vi undersøgte ekspressionsniveauerne af miRNA'er i nyrerne hos HIGA-mus (n = 10). Dette resultat blev opnået fuldstændigt baseret på den beskrevne protokol. Balb/c-musenes nyrer blev valgt som kontrol (n=10). I begge grupper blev der udvalgt 25 uger gamle. Kun kvindelige HIGA-mus var tilgængelige fra leverandøren. Ekspressionsniveauerne for tre miRNA'er (miR-155-5p, miR-146a-5p og miR-21-5p; Figur 1) blev påvist, som tidligere blev rapporteret at være forbundet med IgA nefropati<sup ...

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

microRNA'er er involveret i patogenesen af IgA nefropati. Vi har udviklet en pålidelig metode til påvisning af microRNA-ekspressionsniveauer i nyrerne hos en IgA-nefropati-musemodel (HIGA-mus). Denne nye metode vil gøre det lettere at kontrollere for miRNAs involvering i IgA nefropati.

Påvisning af MicroRNA-ekspression i nyrerne hos immunoglobulin A nefropatiske mus

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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