Name: isolation of small noncoding rnas from human serum
Uploaded: 2014-06-19T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Small Noncoding RNAs from Human Serum at JoVE.com

Samantha Khoury og Pamela Ajuyah understøttes af den australske Postgraduate Award. Vi vil også gerne anerkende den Translationel Cancer Research Network, Lowy Cancer Research Centre, University of New South Wales og Northern Translationel Cancer Research Unit for deres ekstra støtte til Samantha Khoury.

Bemærk: Human serum prøver fra raske patienter eller patienter med cancer blev opnået med informeret samtykke under godkendte humane etiske protokoller fra Royal Prince Alfred Hospital Sydney (Protokol nummer X10-0016 og HREC/10/RPAH/24) og University of Technology, Sydney. Serumprøver blev opsamlet fra patienter før operation fra forskellige Sydney hospitaler og placeres i opbevaring ved -80 ° C. 1. Small RNA Isolation fra serum Totalt RNA blev fremstillet u...

<ol>
	<li>Babu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+extraction+efficiency+of+small+RNAs+with+the+Agilent+2100+Bioanalyzer+and+the+Small+RNA+Kit.">Monitoring extraction efficiency of small RNAs with the Agilent 2100 Bioanalyzer and the Small RNA Kit.</a> Agilent Application Note. , (2010).</li><li>Tran, N., Hutvagner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+and+the+regulation+of+the+maturation+of+miRNAs.">Biogenesis and the regulation of the maturation of miRNAs.</a> Essays Biochem. 54, 17-28 (2013).</li><li>Chim, S. 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=Detection+and+characterization+of+placental+microRNAs+in+maternal+plasma.">Detection and characterization of placental microRNAs in maternal plasma.</a> Clin. Chem. 54, 482-490 (2008).</li><li>Lodes, M. J., 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=Detection+of+cancer+with+serum+miRNAs+on+an+oligonucleotide+microarray.">Detection of cancer with serum miRNAs on an oligonucleotide microarray.</a> PLoS One. 4, (2009).</li><li>Chen, X., 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=Characterization+of+microRNAs+in+serum:+a+novel+class+of+biomarkers+for+diagnosis+of+cancer+and+other+diseases.">Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases.</a> Cell Res. 18, 997-1006 (2008).</li><li>Burgos, K. L., 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=Identification+of+extracellular+miRNA+in+human+cerebrospinal+fluid+by+next-generation+sequencing.">Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing.</a> RNA. 19, 712-722 (2013).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction.">Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.</a> Anal. Biochem. 162, 156-159 (1987).</li><li>Zhang, X., 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=Alterations+in+miRNA+processing+and+expression+in+pleomorphic+adenomas+of+the+salivary+gland.">Alterations in miRNA processing and expression in pleomorphic adenomas of the salivary gland.</a> Int. J. Cancer. 124, 2855-2863 (2009).</li><li>Chomczynski, P., Sacchi, 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+single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction:+twenty-something+years+on.">The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on.</a> Nat. Protoc. 1, 581-585 (2006).</li><li>Kirschner, M. B., 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=Haemolysis+during+sample+preparation+alters+microRNA+content+of+plasma.">Haemolysis during sample preparation alters microRNA content of plasma.</a> PLoS One. 6, (2011).</li><li>Murphy, N. R., Hellwig, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+nucleic+acid+organic+extraction+through+use+of+a+unique+gel+barrier+material.">Improved nucleic acid organic extraction through use of a unique gel barrier material.</a> Biotechniques. 21, 934-936 (1996).</li><li>Taylor, C. J., Satoor, S. N., Ranjan, A. K., Pereira e Cotta, M. V., Joglekar, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+measurement+of+noncoding+RNA+in+human+serum.">A protocol for measurement of noncoding RNA in human serum.</a> Experimental Diabetes Research. 2012, (2012).</li><li>Ichikawa, M., Akiyama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Combination+of+Extraction+Reagent+and+DNA+Microarray+That+Allows+for+the+Detection+of+Global+MiRNA+Profiles+from+Serum/Plasma.">A Combination of Extraction Reagent and DNA Microarray That Allows for the Detection of Global MiRNA Profiles from Serum/Plasma.</a> Methods Mol. Biol. 1024, 247-253 (2013).</li><li>Fromm, B., Harris, P. D., Bachmann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+preparations+from+individual+monogenean+Gyrodactylus+salaris-a+comparison+of+six+commercially+available+totalRNA+extraction+kits.">MicroRNA preparations from individual monogenean Gyrodactylus salaris-a comparison of six commercially available totalRNA extraction kits.</a> BMC Res. Notes. 4, 217 (2011).</li><li>Li, Y., Kowdley, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+microRNA+isolation+from+clinical+serum+samples.">Method for microRNA isolation from clinical serum samples.</a> Anal. Biochem. 431, 69-75 (2012).</li><li>McAlexander, M. A., Phillips, M. J., Witwer, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Methods+for+miRNA+Extraction+from+Plasma+and+Quantitative+Recovery+of+RNA+from+Cerebrospinal+Fluid.">Comparison of Methods for miRNA Extraction from Plasma and Quantitative Recovery of RNA from Cerebrospinal Fluid.</a> Frontiers in Genetics. 4, 83 (2013).</li><li>Kim, Y. K., Yeo, J., Kim, B., Ha, M., Kim, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+structured+RNAs+with+low+GC+content+are+selectively+lost+during+extraction+from+a+small+number+of+cells.">Short structured RNAs with low GC content are selectively lost during extraction from a small number of cells.</a> Mol. Cell. 46, 893-895 (2012).</li></ol>

Populationen af ​​microRNA udgør ca 0,4-0,5% af det totale RNA fundet i serum. Endvidere er der også et højt proteinindhold findes i humant serum. For at forbedre RNA og reducere både protein og phenol forurener vi har ændret den traditionelle Chomczynski tilgang 9 med tilføjelse af flere trin. Totalt RNA blev isoleret fra serum under anvendelse af standard Tri-Reagent RT-LS (Molecular Research Center), men når den udføres i vores laboratorium, er denne metode gav RNA ...

I de senere år har der været en voksende indsats for at opdage nye biomarkører til tidlig påvisning af sygdomme hos mennesker. Meget opmærksomhed har været fokuseret på at bruge små RNA såsom microRNA 2 (miRNA eller Mirs) som potentielle markører. Disse små RNA&#39;er findes i kropsvæsker, såsom serum, og undersøgelser har vist, at de er modstandsdygtige over for nedbrydning og er stabile over et område af varierende miljøforhold 3. På baggrund af disse funktioner, serum-eller cirkulerende miRNA er den ideelle biomarkør 4, 5. I øjeblikket er der to primære metoder til isolering af små RNA fra biologiske væsker. Den første fremgangsmåde bruger kolonne-baseret teknologi til at binde og elueres små RNA 6, mens den anden fremgangsmåde bruger den langvarige protokol med phenol og guanidiniumthiocyanat reagenser 7. Vi har udviklet en enkel, effektiv, kolonne-fri protokol til at isolere små RNA fra humant serum. Det isolerede RNA straksdelbart anvendelige i downstream-applikationer, herunder DNA oligonukleotid arrays og RNA sekventering. Denne protokol blev udviklet, fordi vi blev konfronteret med flere problemer, når du bruger phenol-baserede metoder til at isolere RNA fra serum. Den traditionelle Chomczynski metode anvendes ofte i de fleste laboratorier med en række reagenser tilgængelige fra de fleste kommercielle leverandører. Dog overvejer deres udbredte anvendelse, strenge retningslinjer ikke er blevet udviklet til konsekvent at producere høj kvalitet RNA fra kropsvæsker, især blod eller serum. Almindelige problemer forbundet med at isolere RNA fra serum omfatter lave RNA udbytter og forurening med reagenser, der anvendes i løbet af isolation, især phenol. Vores tilgang eliminerer disse phenol forureninger til at levere kvalitet RNA høj for downstream analyse som kvantitativ PCR (qPCR) og RNA sekventering. Vi har yderligere testet denne RNA på miRNA arrays.

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			<td height="22" style="height:22px;width:259px;">Name of the Material/Equipment&#160;</td>
			<td style="width:223px;">Company&#160;</td>
			<td style="width:123px;">Catalog Number&#160;</td>
			<td style="width:248px;">Comments/ Description (optional)&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Tri-Reagent RT LS</td>
			<td style="width:223px;">Molecular Research Centre, USA</td>
			<td style="width:123px;">TR 118</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:259px;">RNase free H20</td>
			<td style="width:223px;">GIBCO Invitrogen</td>
			<td style="width:123px;">10977-023</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Proteinase K&#160;</td>
			<td style="width:223px;">Finnzymes, Finland</td>
			<td style="width:123px;">EO0491</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Heavy Phaselock Tube</td>
			<td style="width:223px;">5PRIME</td>
			<td>2302830</td>
			<td style="width:248px;">2ml capacity</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">DNA Lobind tube</td>
			<td style="width:223px;">Eppendorf</td>
			<td style="width:123px;">0030 108.078</td>
			<td style="width:248px;">1.5ml capacity</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Glycogen&#160;</td>
			<td style="width:223px;">Invitrogen, USA</td>
			<td style="width:123px;">10814-010</td>
			<td style="width:248px;">5mg/ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">RNA grade Isopropanol&#160;</td>
			<td style="width:223px;">Sigma Aldrich, USA</td>
			<td style="width:123px;">I9516</td>
			<td style="width:248px;">100%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Refrigerated Centrifuge</td>
			<td style="width:223px;">John Morris</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Nanodrop UV-Vis spectrophotometer</td>
			<td style="width:223px;">Thermo Fisher Scientific, USA</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">RNA grade Ethanol</td>
			<td style="width:223px;">Sigma Aldrich, USA</td>
			<td style="width:123px;">E7023</td>
			<td style="width:248px;">70%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Agilent 2100 Bioanalyser</td>
			<td style="width:223px;">Agilent, USA</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
		</tr>
	</tbody>
</table>

isolation of small noncoding rnas from human serum

Analysen af ​​RNA og dets ekspression er et fælles træk i mange laboratorier. Af betydning er fremkomsten af ​​små RNA som microRNA, som findes i pattedyrceller. Disse små RNA er potente gen regulatorer styrer vitale veje såsom vækst, udvikling og død og stor interesse har været rettet på deres udtryk i kropsvæsker. Dette er på grund af deres dysregulering i humane sygdomme som kræft og deres potentielle anvendelse som serum biomarkører. Analyse af miRNA ekspression i serum, kan dog være problematisk. I de fleste tilfælde mængden af serum er begrænsende og serum indeholder lave mængder af total RNA, hvoraf små RNA kun udgør 0,4-0,5% 1. Isolering af tilstrækkelige mængder af kvalitet RNA fra serum er således en stor udfordring for forskerne i dag. I denne tekniske papir, vi demonstrere en metode, som kun bruger 400 ul humant serum for at opnå tilstrækkelig RNA til enten DNA arrays eller qPCR analyse. Den advantages ved denne metode er dens enkelhed og evne til at give kvalitet RNA høj. Det kræver ingen særlige kolonner til rensning af små RNA og udnytter almindelige reagenser og hardware, der findes i almindelige laboratorier. Vores metode anvender en faselåst Gel at eliminere phenol forurening ved samtidig opnåelse af høj kvalitet RNA. Vi introducerer også en yderligere skridt til yderligere at fjerne alle forureninger under isolation trin. Denne protokol er meget effektiv til at isolere udbyttet af total RNA på op til 100 ng / ul fra serum, men kan også tilpasses til andre biologiske væv.

Figur 1 viser et typisk UV / Vis-spektret af RNA isoleret fra serum. Fra denne profil, vi bemærkede protein forurening ved 280 nm med phenol og organiske forureninger både på 270 nm og 230 nm, hhv. Rest guanidiniumthiocyanat blev også bemærket ved 260 nm. For at reducere forurenende stoffer, blev en række optimering skridt foretaget standard Tri-Reagent RT-LS procedure. Vi har tilføjet 5 mg / ml af glykogen at øge både den totale RNA udbytte og reducere forureningen (sort linje, figur 1...

Watch this Scientific Journal Video about Isolation of Small Noncoding RNAs from Human Serum at JoVE.com

Isolation of Small Noncoding RNAs from Human Serum

Denne protokol beskriver en fremgangsmåde til ekstraktion af små RNA&#39;er fra humant serum. Vi har brugt denne metode til at isolere microRNA fra kræft-serum til anvendelse i DNA-arrays og også singleplex kvantitativ PCR. Protokollen anvender phenol og guanidiniumthiocyanat reagenser med modifikationer til opnåelse kvalitet RNA høj.

Isolering af Small noncoding RNA&#39;er fra human serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

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