Name: cell type-specific gene expression profiling in the mouse liver
Uploaded: 2019-09-17T14:30:00
Description: Watch this Scientific Journal Video about Cell Type-specific Gene Expression Profiling in the Mouse Liver at JoVE.com

Dette arbeidet ble støttet av følgende tilskudd: F31-DK113666 (AWW), K01-DK102868 (AMZ), K08-DK106478 (KJW) og P30-DK050306 (pilot stipend til KJW).

Alle metoder som involverer bruk av mus er i samsvar med retningslinjene gitt av IACUC av penn Office of Animal Welfare ved University of Pennsylvania.
1. forberedelse av reagens
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Cycloheximide
	<ol>
<li>For å gjøre 500 μL av 0,1 g/mL cycloheximide, suspendere 50 mg cycloheximide i 500 μL av metanol. Forsiktig: Cycloheximide er ekstremt giftig for miljøet og kan forårsake medfødt misdannelse. Alt avfall og buffere som inneholder cycloheximi...

<ol>
	<li>Taub, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration:+from+myth+to+mechanism.">Liver regeneration: from myth to mechanism.</a> Nature Reviews Molecular Cell Biology. 5 (10), 836-847 (2004).</li><li>Lee, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Etiologies+of+acute+liver+failure.">Etiologies of acute liver failure.</a> Seminars in Liver Disease. 28 (2), 142-152 (2008).</li><li>Sanyal, A. J., Yoon, S. K., Lencioni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+etiology+of+hepatocellular+carcinoma+and+consequences+for+treatment.">The etiology of hepatocellular carcinoma and consequences for treatment.</a> The Oncologist. 15 Suppl 4, 14-22 (2010).</li><li>Jadlowiec, C. C., Taner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+transplantation:+Current+status+and+challenges.">Liver transplantation: Current status and challenges.</a> World Journal of Gastroenterology. 22 (18), 4438-4445 (2016).</li><li>Michalopoulos, G. K., DeFrances, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration.">Liver regeneration.</a> Science. 276 (5309), 60-66 (1997).</li><li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration+after+partial+hepatectomy:+critical+analysis+of+mechanistic+dilemmas.">Liver regeneration after partial hepatectomy: critical analysis of mechanistic dilemmas.</a> The American Journal of Pathology. 176 (1), 2-13 (2010).</li><li>Grompe, M., 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=Loss+of+fumarylacetoacetate+hydrolase+is+responsible+for+the+neonatal+hepatic+dysfunction+phenotype+of+lethal+albino+mice.">Loss of fumarylacetoacetate hydrolase is responsible for the neonatal hepatic dysfunction phenotype of lethal albino mice.</a> Genes &amp; Development. 7 (12A), 2298-2307 (1993).</li><li>Wangensteen, K. 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=A+facile+method+for+somatic,+lifelong+manipulation+of+multiple+genes+in+the+mouse+liver.">A facile method for somatic, lifelong manipulation of multiple genes in the mouse liver.</a> Hepatology. 47 (5), 1714-1724 (2008).</li><li>Wang, A. W., 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=TRAP-seq+identifies+cystine/glutamate+antiporter+as+a+driver+of+recovery+from+liver+injury.">TRAP-seq identifies cystine/glutamate antiporter as a driver of recovery from liver injury.</a> The Journal of Clinical Investigation. 128 (6), 2297-2309 (2018).</li><li>Heiman, M., Kulicke, R., Fenster, R. J., Greengard, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+mRNA+purification+by+translating+ribosome+affinity+purification+(TRAP).">Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP).</a> Nature Protocols. 9 (6), 1282-1291 (2014).</li><li>Agilent Technologies. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Agilent RNA 6000 Pico: User Manual. , (2016).</li><li>NEBNext. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NEBNext Ultra RNA Library Prep Kit for Illumina: User Manual. , (2018).</li><li>NanoDrop. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2000/2000c Spectrophotomerter: User Manual. , (2009).</li><li>Liu, 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=Cell-specific+translational+profiling+in+acute+kidney+injury.">Cell-specific translational profiling in acute kidney injury.</a> The Journal of Clinical Investigation. 124 (3), 1242-1254 (2014).</li><li>Lu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glutathione+synthesis.">Regulation of glutathione synthesis.</a> Molecular Aspects of Medicine. 30 (1-2), 42-59 (2009).</li><li>Wang, A. W., 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+dynamic+chromatin+architecture+of+the+regenerating+liver.">The dynamic chromatin architecture of the regenerating liver.</a> bioRxiv. , (2019).</li><li>Zahm, A. M., 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=A+high-content+in+vivo+screen+to+identify+microRNA+epistasis+in+the+repopulating+mouse+liver.">A high-content in vivo screen to identify microRNA epistasis in the repopulating mouse liver.</a> bioRxiv. , (2019).</li><li>Stork, C., Zheng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Profiling+of+RNA-Protein+Interactions+Using+CLIP-Seq.">Genome-Wide Profiling of RNA-Protein Interactions Using CLIP-Seq.</a> Methods in Molecular Biology. 1421, 137-151 (2016).</li><li>Zhao, 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=Glutathione+antioxidant+pathway+activity+and+reserve+determine+toxicity+and+specificity+of+the+biliary+toxin+biliatresone+in+zebrafish.">Glutathione antioxidant pathway activity and reserve determine toxicity and specificity of the biliary toxin biliatresone in zebrafish.</a> Hepatology. 64 (3), 894-907 (2016).</li><li>Halpern, K. 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=Single-cell+spatial+reconstruction+reveals+global+division+of+labour+in+the+mammalian+liver.">Single-cell spatial reconstruction reveals global division of labour in the mammalian liver.</a> Nature. 542 (7641), 352-356 (2017).</li><li>Camp, J. 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=Multilineage+communication+regulates+human+liver+bud+development+from+pluripotency.">Multilineage communication regulates human liver bud development from pluripotency.</a> Nature. 546 (7659), 533-538 (2017).</li><li>Wang, Y. J., Kaestner, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+RNA-Seq+of+the+Pancreatic+Islets--a+Promise+Not+yet+Fulfilled?.">Single-Cell RNA-Seq of the Pancreatic Islets--a Promise Not yet Fulfilled?.</a> Cell Metabolism. 29 (3), 539-544 (2019).</li></ol>

FELLE-SEQ er en teknikk for cellen type-spesifikk isolasjon av oversette mRNA via epitope-merket ribosomer og gaver en alternativ å FACS tilnærmelser, for den omgår begrensninger som tid behov av FACS9. I stedet tillater TRAP rask og effektiv isolering av RNA direkte fra masse vev, noe som bidrar til å unngå eventuelle endringer i genuttrykk. Trap-SEQ er spesielt velegnet for bruk i repopulating Fah-/- mus leveren, som hepatocytter ekspansjon etter fjerning av nitisinone ...

Som de viktigste metabolske organ i virveldyr, leveren er ansvarlig for glukose homeostase, serum proteinsyntese, galle syre sekresjon, og xenobiotic oval metabolisme og avgiftning. Leveren besitter en ekstraordinær evne til å regenerere den skadde parenchyma upon eksponering for giftstoffer for å hindre umiddelbar leversvikt1. Imidlertid kan svikt i regenerering oppstå i innstillingen av paracetamol eller alkohol overforbruk, som kan føre til akutt leversvikt2. Videre, kronisk leverskader forårsaket av viral hepatitt infeksjon, fatty leversykdom, og steatohepatitis kan forårsake leveren fibrose, skrumplever, og leverkreft3. Den eneste tilgjengelige helbredende behandlingen for sluttfasen leversykdom er transplantasjon, men er begrenset av organ mangel, og forhindrer effektiv behandling for alle pasienter4. En bedre forståelse av gjenopprettingsprosessen etter giftig leverskade er derfor avgjørende for utvikling av behandlinger for å stimulere regenerering tilstrekkelig til å redde funksjon i det syke orgelet.
Den mest omfattende anvendt modell system for studiet av leveren gjenfødelse er delvis hepatectomy i gnagere, der en stor andel av leveren er resected å stimulere rask hepatocytter ekspansjon5. Men, delvis hepatectomy ikke recapitulate hepatocytter ekspansjon etter giftig leverskade på grunn av mangel på immun celle infiltrasjon og hepatocytter celle nekrose ofte observert i innstillingen av akutt leverskade hos mennesker6. Et mer egnet system for å modellere denne formen for orgel fornyelse er Fah-/- mus, som mangler funksjonell fumarylacetoacetate Hydrolase (Fah) kreves for riktig tyrosin metabolisme og utvikler alvorlig leverskader fører til død7. Disse musene kan opprettholdes i en sunn tilstand på ubestemt tid ved behandling med stoffet nitisinone i drikkevannet. Alternativt kan FAH uttrykk gjenopprettes ved transgene levering til en undergruppe av hepatocytter, som vil utvide til gjenbefolke leveren ved nitisinone fjerning8.
Å profil genuttrykk endringer av repopulating hepatocytter, et verktøy for å spesifikt isolere replikere hepatocytter i Fah-/- mus uten forurensning fra nabolandet skadde hepatocytter og andre celletyper er nødvendig. Dessverre er fluorescens-assistert celle sortering (FACS) av hepatocytter vanskelig siden (1) skjørhet av repopulating hepatocytter fører til dårlig restitusjon etter leveren, (2) replikere hepatocytter er svært variabel i størrelse, noe som gjør isolasjon av en ren befolkning av FACS vanskelig, og (3) prosedyren tid fra leveren til RNA isolasjon er større enn 2 h, derav genuttrykk profiler kan gjennomgå betydelige kunstige endringer før prøvene er ervervet9.
Alternativt, uttrykk for epitope-merket ribosomer spesielt i repopulating hepatocytter tillater rask isolering av aktivt oversette mRNA bundet av ribosomer ved hjelp av affinitet rensing umiddelbart etter orgel innhøsting, med bulk lever vev lysater. Her beskriver vi en protokoll for å utføre oversette-ribosom affinitet rensing (TRAP)10 etterfulgt av høy gjennomstrømming RNA-SEKVENSERING (Trap-SEQ), til spesielt isolere og profil mRNA i repopulating hepatocytter i Fah-/- mus9. Coexpression av grønne fluorescerende protein-merket ribosomal protein (GFP: RPL10A) med FAH tillater affinitet rensing av oversette mRNA bundet av polysomes som inneholder GFP: RPL10A. Denne metoden unngår alle celle dissosiasjon trinn, for eksempel lever-og repopulating hepatocytter. I stedet utnytter det lyse av hele orgel vev og antistoffer for å raskt trekke ut RNA spesielt fra målet cellene. Til slutt, isolering av rikelig, høy kvalitet mRNA via TRAP-SEQ muliggjør nedstrøms applikasjoner som sekvensering analyse til profilen den dynamiske endringen av genet uttrykk under Repopulation prosessen.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Tissue Grinder, Potter-Elv, Coated</td>
			<td>DWK Life Sciences (Wheaton)</td>
			<td>358007</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolutely RNA Miniprep Kit</td>
			<td>Agilent</td>
			<td>400800</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP antibodies</td>
			<td>Memorial Sloan-Kettering Antibody &#38; Bioresource Core</td>
			<td colspan="2">GFP Ab #19C8 and GFP Ab #19F7</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, IgG-Free, Protease-Free</td>
			<td>Jackson ImmunoResearch</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Millipore Sigma</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxycholic acid, DOC</td>
			<td>Millipore Sigma</td>
			<td>D2510</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose, Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Millipore Sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads MyOne Streptavidin T1</td>
			<td>Thermo Fisher Scientific</td>
			<td>65602</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Petri Dishes with Clear Lid</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (10x), calcium, magnesium, no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>14065-056</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, 1M Solution, pH 7.3, Molecular Biology Grade, Ultrapure, Thermo Scientific</td>
			<td>Thermo Fisher Scientific</td>
			<td>AAJ16924AE</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride, MgCl2&#160;</td>
			<td>Millipore Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000/2000c Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>VV-83061-00</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext Poly(A) mRNA Magnetic Isolation Module</td>
			<td>New England BioLabs</td>
			<td>E7490S</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext Ultra RNA Library Prep Kit for Illumina</td>
			<td>New England BioLabs</td>
			<td>E7530S</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonylphenyl polyethylene glycol, NP-40. IGEPAL CA-630</td>
			<td>Millipore Sigma</td>
			<td>I8896</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water, not DEPC-Treated</td>
			<td>Ambion</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>Overhead Stirrer</td>
			<td>DWK Life Sciences (Wheaton)</td>
			<td>903475</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Buffer (10x), pH 7.4</td>
			<td>Ambion</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Recombinant Protein L, Biotinylated</td>
			<td>Thermo Fisher Scientific</td>
			<td>29997</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride, KCl</td>
			<td>Millipore Sigma</td>
			<td>P4504</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA 6000 Pico Kit &#38; Reagents</td>
			<td>Agilent</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseZap RNase Decontamination Solution</td>
			<td>Invitrogen</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin Ribonuclease Inhibitors</td>
			<td>Promega</td>
			<td>N2515</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide, NaN3</td>
			<td>Millipore Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate, NaHCO3</td>
			<td>Millipore Sigma</td>
			<td>S6297</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPERase&#183;In RNase Inhibitor</td>
			<td>Invitrogen</td>
			<td>AM2694</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell type-specific gene expression profiling in the mouse liver

Liver Repopulation etter skade er en viktig funksjon av pattedyr som hindrer umiddelbar organ svikt og død etter eksponering av miljøgifter. En dypere forståelse av endringene i genuttrykk som oppstår under Repopulation kan bidra til å identifisere terapeutiske mål for å fremme restaurering av leverfunksjon i innstillingen av skader. Likevel, metoder for å isolere spesielt de repopulating hepatocytter er hemmet av mangel på celle markører, begrenset celle tall, og skjørhet av disse cellene. Utviklingen av oversette ribosom affinitet rensing (Trap) teknologi i forbindelse med Fah-/- Mouse modell for å recapitulate Repopulation i innstillingen av leverskade tillater genuttrykk profilering av repopulating hepatocytter. Med TRAP, celle type-spesifikke oversette mRNA er raskt og effektivt isolert. Vi utviklet en metode som utnytter TRAP med affinitet-baserte isolering av oversette mRNA fra hepatocytter som selektivt uttrykker det grønne fluorescerende proteinet (GFP)-Tagged ribosomal protein (RP), GFP: RPL10A. TRAP omgår den lange tidsperioden som kreves for fluorescens celle sortering som kan endre gen uttrykks profilen. Videre, siden bare repopulating hepatocytter uttrykke GFP: RPL10A Fusion protein, den isolerte mRNA er blottet for forurensning fra omkringliggende skadde hepatocytter og andre celletyper i leveren. Affinitet-renset mRNA er av høy kvalitet og tillater nedstrøms PCR-eller høy-gjennomstrømning sekvensering-basert analyse av genuttrykk.

Til profil genuttrykk i repopulating hepatocytter av Fah-/- Mouse, Gfp: Rpl10a Fusion og Fah transgenes er co-levert i en Transposon-inneholdende plasmider8 (Trap Vector) til lever av hydrodynamisk injeksjon (figur 1A). Fjerning av nitisinone induserer en giftig leverskade som skaper et utvalg press for hepatocytter stabilt uttrykker FAH å gjenbefolke den skadde parenchyma<sup cla...

Watch this Scientific Journal Video about Cell Type-specific Gene Expression Profiling in the Mouse Liver at JoVE.com

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Oversette ribosom affinitet rensing (TRAP) muliggjør rask og effektiv isolering av celle type-spesifikke oversette mRNA. Her viser vi en metode som kombinerer hydrodynamisk hale-blodåre injeksjon i en mus modell av leveren Repopulation og TRAP å undersøke uttrykket profilen til repopulating hepatocytter.

Cell type-spesifikke Gene Expression profilering i musen Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Med TRAP-seq kan vi isolere RNA fra bestemte celletyper, for eksempel hepatocytter som repopulerer i skadet lever for å analysere genuttrykksendringene som forekommer i disse cellene. Det krever ingen trinn for å fjerne uønskede celler fra vevet. Celletypespesifikke RNA er isolert ved affinitetsrensing fra hele orgellylater.Denne teknikken hjelper oss med å avgjøre hvordan spesifikke celler reagerer på skader, noe som kan bidra til å identifisere nye strategier eller legemidler for å bekjempe leversykdom. Dette kan brukes til å identifisere hvordan leverceller reagerer på ulike typer leverskader. Foreløpig er det få alternativer for alvorlige akutte leverskader, og denne metoden vil bidra til å gi oss en ledetråd til nye behandlinger.Denne metoden oppsto i hjernen. I leveren så vi for oss at den kunne brukes til å undersøke dynamiske endringer i en rekke celletyper, hepatocytter, gallekanalceller, Kupffer-celler og endotelceller for å nevne noen. Å demonstrere prosedyren vil være Amber Wang, en utdannet student og min mentee.For å begynne, resuspend magnetiske perler ved mild pipettering. Samle perlene på et magnetisk stativ i mer enn ett minutt og fjern det overnaturlige. Fjern deretter mikrocentrifugerøret fra magnetstativet og tilsett en milliliter PBS, etterfulgt av pipettering opp og ned for å vaske perlene.Plasser røret tilbake på magnetstativet i mer enn ett minutt for å samle perlene og fjerne PBS. For å forberede protein L-belagte perler, tilsett 16 mikroliter biotinyler protein L til de resuspenderte og vasket magnetiske perler og tilsett 1X PBS for å gjøre det endelige volumet en milliliter, hvis du bruker et 1,5-milliliter mikrocentrifugerør, eller 1,5 milliliter, hvis du bruker et to milliliter mikrocentrifugerør. Inkuber de magnetiske perlene med biotinylert protein L i 35 minutter ved romtemperatur på en rørrotator.Deretter plasserer du røret på magnetstativet i mer enn ett minutt og fjerner det overnaturlige for å samle proteinet L-belagte perler. Fjern mikrocentrifugerøret fra magnetstativet og tilsett en milliliter PBS med 3% BSA-buffer, etterfulgt av mild pipettering minst fem ganger for å vaske proteinet L-belagte perler. Igjen, plasser røret på magnetstativet i mer enn ett minutt og fjern supernatanten for å samle de bebelagte perlene.Gjenta BSA vask ytterligere fire ganger. Deretter legger du til 50 mikrogram antistoffer for GFP antistoff 19C8 og GFP antistoff 19F7 hver inn i proteinet L-belagte perler, og inkubere i en time ved fire grader Celsius på en rørrotator. For å samle affinitetsmatrisen, legg røret på magnetstativet i mer enn ett minutt og fjern det overnaturlige.Ta røret bort fra magnetstativet og tilsett en milliliter med lavsaltbuffer. Rør forsiktig opp og ned for å vaske affinitetsmatrisen og gjenta lavsaltbufferen som vasker ytterligere to ganger. Resuspend perlene i 200 mikroliter med lav-salt buffer, for å lage 200 mikroliter affinitet matrise.Først setter du opp vevskvernen slik at PTFE-glassrørene kan plasseres på is under homogenisering. Fyll fire milliliter kald lysisbuffer i PTFE-glassrørene. Legg leveren på en petriskål.Bruk en kniv til å isolere 200 til 500 milligram leverbiter og flytte inn i PTFE glassrør. Plasser gjenværende levervev i et forkjølet mikrocentrifugerør og blits fryse i flytende nitrogen. Homogenisere prøvene i en motordrevet homogenisator, starter på 300 rpm for minst fem slag for å fjerne hepatocytter fra leverstrukturen.Senk glassrøret hver gang. Forhindre øl, noe som kan forårsake proteindening, ved å holde pestle under løsningen. Deretter øker du hastigheten til 900 rpm for å fullstendig homogenisere levervevet i minst 12 fulle slag.Overfør lysatet til merkede og forkjølte rør med ikke mer enn en milliliter lysate hver. For å starte kjernefysisk lysis, sentrifugere leveren lysate på 2, 000 ganger g ved fire grader Celsius i 10 minutter, og deretter overføre supernatant til en ny pre-kjølt mikrocentrifuge tube på is. Tilsett 1/9th av det overnaturlige volumet på 10% NP-40 i mikrocentrifugerøret på is og bland løsningen ved å forsiktig invertere røret.Spinn raskt ned mikrocentrifugerørene med en mini sentrifuge og tilsett 1/9th av prøvevolumet på 10% DOC. Bland ved å forsiktig invertere mikrocentrifugerørene og raskt spinne ned mikrocentrifugerørene og inkubere på is i fem minutter. Sentrifuger atomlysatet på 20 000 ganger g ved fire grader Celsius i 10 minutter og overfør supernatanten til nye prekjølte mikrocentrifugerør på is.For hvert rør, ta ut 1% av det totale volumet av supernatant som en pre-immunoprecipitation kontroll. Plasser pre-immunoprecipitation kontrollene på en rør rotator på fire grader Celsius over natten. Vær ekstra forsiktig for å forsiktig bruke affinitetsmatrisen ved å pipettering, og legg deretter til 200 mikroliter i hver prøve.Inkuber lysates på fire grader Celsius over natten med mild blanding på en rør rotator. For å isolere RNA, snurr raskt ned rørene som inneholder lysates i mini sentrifuge og samle perlene ved å plassere på magnetstativet i minst ett minutt. Samle supernatant i ekstra mikrocentrifuge rør.Tilsett en milliliter fersk høysaltbuffer til hvert rør, etterfulgt av skånsom pipettering minst fem ganger uten å introdusere bobler. Samle perlene på det magnetiske stativet på is i over ett minutt og fjern det overnaturlige. Gjenta vasketrinnene ytterligere fire ganger.Fjern deretter mikrocentrifugerørene fra magnetstativet og plasser ved romtemperatur i fem minutter for å varme opp. Resuspend perlene i 100 mikroliter lysis buffer med 0,7 mikroliter beta-mercaptoetanol for å frigjøre bundet RNA fra affinitetsmatrisen. Vortex rørene i minst fem sekunder med høyeste hastighet og raskt spinne ned.Deretter inkubere rørene ved romtemperatur i 10 minutter. Plasser rørene på magnetstativet i minst ett minutt, samle deretter supernatanten og fortsett umiddelbart til RNA-opprydding i henhold til RNA-renseprotokollen i settet. For å oppnå maksimal kvalitet på den isolerte RNA, utfør alle valgfrie trinn, inkludert DNase fordøyelse og alle RNA elution trinn, inkludert anbefalt forvarming av elution buffer til 60 grader Celsius.I denne studien ble GFP-L10A fusjon og Fah transgener levert i en transposonholdig plasmidfellevektor til lever ved hydrodynamisk injeksjon. Fjerning av nitisinin induserte en giftig leverskade. Immunofluorescence farging bekreftet samtidiguttrykk av FAH og GFP-L10A fusjonprotein og repopulating hepatocytter etter to uker med lever repopulation.Den quiescent prøven produsert det høyeste utbyttet av fusjon protein, siden alle hepatocytter uttrykke GFP-L10A etter AAV8-TBG-Cre injeksjon. Omvendt var knapt noen RNA påviselig i wild-type kontroller som ikke hadde GFP-L10A transgene, noe som indikerer trap prosedyren er svært spesifikk og har lav bakgrunn. Når TRAP ble brukt på levervev gjennomgår repopulation med GFP-L10A transdusert hepatocytter, rikelig høy kvalitet RNA ble oppnådd.I motsetning ble det ikke oppdaget RNA-spor via Bioanalyzer for den negative kontrollprøven. Gsta1-uttrykket ble indusert av over tidoblet i repopulating hepatocytter sammenlignet med quiescent hepatocytter, mens ingen terskelsyklusverdier ble oppdaget med TRAP-isolert RNA fra villtypemusen på grunn av mangel på inngang RNA. Når du homogeniserer vevet, sørg for å bevege pestle sakte for å forhindre aerasjon.Etter å ha isolert RNA, kan høy gjennomstrømningssekvensering eller qPCR utføres for å analysere genuttrykk. Med TRAP-seq har vi nå en metode for å isolere RNA spesifikt fra å reponere hepatocytter og analysere endringen i genuttrykk under leverrebefolkning. Dette gjør at vi kan identifisere gener som endres under denne prosessen og potensielle terapeutiske mål for behandling av leverskade.Cykloheximid og DTT, som er tilstede i flere buffere, er begge giftige. Avfall skal samles inn og kastes i henhold til institusjonelle retningslinjer.

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Molecular Biology

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Unit 1

Gene Expression

SCIENTISTS IN ACTION

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Heterokaryon Technique for Analysis of Cell Type-specific Localization

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling. These proteins display a diverse array of cellular functions including nuclear import/export of RNA and protein, transcriptional regulation, and apoptosis. Interestingly, major cellular reorganizations including cell division, differentiation and transformation, often involve such activities3,4,8,10. The detailed study of these proteins and their respective regulatory mechanisms can be challenging as the stimulation for these localization changes can be elusive, and the movements themselves can be quite dynamic and difficult to track. Studies involving cellular oncogenesis, for example, continue to benefit from understanding pathways and protein activities that differ between normal primary cells and transformed cells6,7,11,12. As many proteins show altered localization during transformation or as a result of transformation, methods to efficiently characterize these proteins and the pathways in which they participate stand to improve the understanding of oncogenesis and open new areas for drug targeting.
Here we present a method for the analysis of protein trafficking and shuttling activity between primary and transformed mammalian cells. This method combines the generation of heterokaryon fusions with fluorescence microscopy to provide a flexible protocol that can be used to detect steady-state or dynamic protein localizations. As shown in Figure 1, two separate cell types are transiently transfected with plasmid constructs bearing a fluoroprotein gene attached to the gene of interest. After expression, the cells are fused using polyethylene glycol, and protein localizations may then be imaged using a variety of methods. The protocol presented here is a fundamental approach to which specialized techniques may be added.

A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

Metabolsk Merking av ny-transkriberte RNA for High Resolution Gene Expression Profilering av RNA syntese, bearbeiding og Decay i cellekultur

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...