Name: detection of microrna expression in peritoneal membrane of rats using quantitative real-time pcr
Uploaded: 2017-06-27T14:00:00
Description: Watch this Scientific Journal Video about Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR at JoVE.com

Vi vil gerne takke Miyako Shigeta for hendes fremragende teknisk support. Dette arbejde blev delvist støttet af JSPS KAKENHI (bevillingsnummer 25461252).

Alle dyreforsøgsprotokoller blev godkendt af dyreetiske udvalg ved Jichi Medical University og blev udført i overensstemmelse med retningslinjerne for anvendelse og pleje af eksperimentelle dyr fra Jichi Medical University Guide for Laboratory Animals. 1. Peritoneumprøveindsamling <ol><li> Saml følgende emner: 50 ml centrifugerør med bomuld drenket i isofluran, korkplade, petriskål med fosfatbufret saltvand (PBS), kirurgiske saks og tang. </li><li> Euthanize en rotte med en overdosis...

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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> Nat Methods. 11 (8), 809-815 (2014).</li><li>Bustin, S. A., 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=The+MIQE+guidelines:+minimum+information+for+publication+of+quantitative+real-time+PCR+experiments.">The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.</a> Clin Chem. 55 (4), 611-622 (2009).</li><li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+real-time+PCR+data+by+the+comparative+C(T)+method.">Analyzing real-time PCR data by the comparative C(T) method.</a> Nat Protoc. 3 (6), 1101-1108 (2008).</li><li>Krediet, R. T., Lindholm, B., Rippe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+peritoneal+membrane+failure.">Pathophysiology of peritoneal membrane failure.</a> Perit Dial Int. 20, S22-S42 (2000).</li><li>Devuyst, O., Margetts, P. J., Topley, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+of+the+peritoneal+membrane.">The pathophysiology of the peritoneal membrane.</a> J Am Soc Nephrol. 21 (7), 1077-1085 (2010).</li><li>Hirahara, I., Ishibashi, Y., Kaname, S., Kusano, E., Fujita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylglyoxal+induces+peritoneal+thickening+by+mesenchymal-like+mesothelial+cells+in+rats.">Methylglyoxal induces peritoneal thickening by mesenchymal-like mesothelial cells in rats.</a> Nephrol Dial Transplant. 24 (2), 437-447 (2009).</li><li>Dallas, P. 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Ved anvendelse af protokollen, der er præsenteret i dette manuskript, blev miRNA&#39;er i rotteperitoneal membran renset med succes og detekteret under anvendelse af qRT-PCR. Pålideligheden af ​​qRT-PCR-dataanalyse afhænger af kvaliteten af ​​rensede miRNA&#39;er. Derfor kan renheden af ​​miRNA&#39;er kontrolleres før qRT-PCR ved forholdet mellem absorbans ved 260 nm og det ved 280 nm, der kan måles under anvendelse af et spektrofotometer. Når signifikant amplifikation af miRNA ikke kan opnås ved anve...

MicroRNA&#39;er (miRNA&#39;er) er korte, ikke-kodende RNA&#39;er, som post-transkriptionelt regulerer messenger-RNA (mRNA) -ekspression 1 . Ændringer i udtrykket af miRNA regulerer ekspressionen af ​​mange mRNA&#39;er, der spiller pivotale roller i forskellige patologiske tilstande, herunder kræft, betændelse, metaboliske forstyrrelser og fibrose 2 , 3 , 4 , 5 , 6 , 7 , 8 . Derfor har miRNA&#39;er potentiale som nye biomarkører og terapeutiske mål 2 , 3 , 4 , 5 , 6 , 7 , 8 . MiRNA ekspressionsprofilen er blevet bestemt i forskellige rotterOrganer og væv, herunder lever, hjerte, lunge og nyre 9 . Standardmetoder til oprensning og påvisning af miRNA&#39;er i rotteperitoneal membran er imidlertid ikke veletablerede. Det overordnede mål med denne protokol er at med succes rense og detektere miRNA&#39;er i rotteperitoneal membranen. For det første blev peritonealmembranprøven homogeniseret ved anvendelse af en glashomogenisator efterfulgt af eksponering for et biopolymer-nedbrydningssystem i en mikrocentrifugespaltkolonne 10 . Dernæst blev total RNA inklusive miRNA oprenset fra peritonealmembranprøven under anvendelse af en silicium-membranbaseret spin-søjle 10 . Derefter blev cDNA syntetiseret fra det oprensede totale RNA under anvendelse af revers transkriptase, poly (A) polymerase og oligo-dT primer 11 . Endelig blev udtrykket af miRNA bestemt ved hjælp af qRT-PCR under anvendelse af et interkalerende farvestof 11 . Begrundelsen for denne protokol er bAsed på tidligere undersøgelser, der viste signifikant oprensning og påvisning af miRNA i væv ved en simpel proces 8 , 10 , 11 . Det er blevet rapporteret, at anvendelsen af ​​et biopolymer-shredding-system i en mikropentrifugespaltkolonne og silikamembranbaseret spin-søjle kan rense højkvalitets total RNA fra væv 10 . Fremgangsmåden til syntetisering af cDNA fra oprenset total RNA under anvendelse af revers transkriptase, poly (A) polymerase og oligo-dT primer og fremgangsmåden til påvisning af miRNA ekspression ved hjælp af qRT-PCR under anvendelse af interkalerende farvestof i denne protokol er blevet rapporteret at vise høj nøjagtighed og Følsomhed 11 . Derudover er dette en simpel proces, hvilket sparer tid og forhindrer teknisk fejl. Derfor er denne protokol nyttig i studier, der kræver meget præcis og følsom påvisning af miRNA i rotteperitoneal membran i en bred vifte af patologiske tilstande.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>QIA shredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>biopolymer-shredding system in a micro centrifuge spin-column</td>
		</tr>
		<tr>
			<td>miRNeasy Mini kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td>silica-membrane based spin column</td>
		</tr>
		<tr>
			<td>QIAzol Lysis Reagent</td>
			<td>Qiagen</td>
			<td>79306</td>
			<td>phenol/guanidine-based lysis reagent</td>
		</tr>
		<tr>
			<td>Buffer RLT</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td>wash buffer 1</td>
		</tr>
		<tr>
			<td>Buffer RWT</td>
			<td>Qiagen</td>
			<td>1067933</td>
			<td>wash buffer 2</td>
		</tr>
		<tr>
			<td>miScript II RT kit&#160;</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td>includes 10&#215; Nucleics Mix containing deoxynucleotides, ribonucleotide triphosphates, and oligo-dT primers; miScript Reverse Transcriptase Mix containing poly(A) polymerase and reverse transcriptase and; miScript HiSpec buffer</td>
		</tr>
		<tr>
			<td>miScript SYBR Green PCR kit</td>
			<td>Qiagen</td>
			<td>218073</td>
			<td>includes QuantiTect SYBR Green PCR Master Mix and miScript Universal Primer</td>
		</tr>
		<tr>
			<td>RNU6-2 primer&#160;&#160;</td>
			<td>Qiagen</td>
			<td>MS00033740</td>
			<td>not disclosed</td>
		</tr>
		<tr>
			<td>miRNA-142-3p primer</td>
			<td>Qiagen</td>
			<td>MS00031451</td>
			<td>5&#39;-UGUAGUGUUUCC 
			UACUUUAUGGA-3&#39;&#160;&#160;</td>
		</tr>
		<tr>
			<td>miRNA-21-5p primer</td>
			<td>Qiagen</td>
			<td>MS00009079</td>
			<td>5&#39;-UAGCUUAUCAG 
			ACUGAUGUUGA-3&#39;</td>
		</tr>
		<tr>
			<td>miRNA-221-3p primer</td>
			<td>Qiagen</td>
			<td>MS00003857</td>
			<td>5&#39;-AGCUACAUUGU 
			CUGCUGGGUUUC-3&#39;</td>
		</tr>
		<tr>
			<td>miRNA-223-3p primer</td>
			<td>Qiagen</td>
			<td>MS00033320</td>
			<td>5&#39;-UGUCAGUUUG 
			UCAAAUACCCC-3&#39;</td>
		</tr>
		<tr>
			<td>miRNA-34a-5p primer</td>
			<td>Qiagen</td>
			<td>MS00003318</td>
			<td>5&#39;-UGGCAGUGUCU 
			UAGCUGGUUGU-3&#39;</td>
		</tr>
		<tr>
			<td>miRNA-327 primer</td>
			<td>Qiagen</td>
			<td>MS00000805</td>
			<td>5&#39;-CCUUGAGGGG 
			CAUGAGGGU-3&#39;</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&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311971</td>
			<td>adhesive film for 96-well reaction plate</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>methylglyoxal</td>
			<td>Sigma-Aldrich</td>
			<td>M0252</td>
			<td></td>
		</tr>
		<tr>
			<td>Midperic</td>
			<td>Terumo</td>
			<td>not assign&#160;</td>
			<td>peritoneal dialysis fluid</td>
		</tr>
		<tr>
			<td>Sprague&#8211;Dawley rats</td>
			<td>SLC</td>
			<td>not assign&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of microrna expression in peritoneal membrane of rats using quantitative real-time pcr

MicroRNA&#39;er (miRNA&#39;er) er små ikke-kodende RNA&#39;er, som regulerer messenger-RNA-ekspression efter transskriptionelt. MiRNA ekspressionsprofilen er blevet undersøgt i forskellige organer og væv i rotter. Standardmetoder til rensning af miRNA&#39;er og påvisning af deres ekspression i rotteperitoneal membran har imidlertid ikke været veletablerede. Vi har udviklet en effektiv og pålidelig metode til at rense og kvantificere miRNA&#39;er ved hjælp af kvantitativ revers-transkriptionspolymerase kædereaktion (qRT-PCR) i rotte peritoneal membran i realtid. Denne protokol består af fire trin: 1) oprensning af peritoneal membranprøve; 2) oprensning af totalt RNA inklusive miRNA fra peritoneal membranprøve; 3) revers transkription af miRNA til fremstilling af cDNA; Og 4) qRT-PCR til påvisning af miRNA-ekspression. Ved hjælp af denne protokol fastslog vi med succes, at udtrykket af seks miRNA&#39;er (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327 og miRNA-34a-5p) stegSignifikant i peritonealmembranen af ​​en peritoneal fibrose-model af rotte sammenlignet med dem i kontrolgrupper. Denne protokol kan bruges til at studere profilen af ​​miRNA-ekspression i peritonealmembranen hos rotter under mange patologiske tilstande.

De resultater, der præsenteres her, er baseret på vores tidligere rapporterede undersøgelse 8 . Vi undersøgte miRNA ekspressionsprofilen i peritoneal fibrose. Peritoneal fibrose er en vigtig komplikation ved peritoneal dialyse. Det er karakteriseret ved tab af mesothelialcelle-monolaget og den overskydende akkumulering af ekstracellulære matrixkomponenter og er forbundet med peritoneal membranfejl 14 , 15</...

Watch this Scientific Journal Video about Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR at JoVE.com

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

Her præsenterer vi en protokol til påvisning af mikroRNA-ekspression i rotte-peritoneal membran ved anvendelse af kvantitativ revers-transkriptionspolymerase-kædereaktion i realtid. Denne metode er egnet til at studere mikroRNA ekspressionsprofilen i rotte peritoneal membran i flere patologiske tilstande.

Påvisning af mikroRNA ekspression i peritoneal membran af rotter ved hjælp af kvantitativ real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

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