Name: combined dna-rna fluorescent in situ hybridization (fish) to study x chromosome inactivation in differentiated female mouse embryonic stem cells
Uploaded: 2014-06-14T14:00:00
Description: Watch this Scientific Journal Video about Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells at JoVE.com

The authors thank all previous and present members of the department of Reproduction and Development of the Erasmus MC for helpful and stimulating discussions during the past years. We also want to thank the three anonymous reviewers for providing helpful feedback. Work in the Gribnau lab is supported by funding from the Dutch research council (NWO-VICI) and an ERC starting grant.

1. Induktion av differentiering i Kvinna embryonala stamceller att inducera X kromosom inaktivering OBS: Kvinnor musen embryonala stamceller (på begäran) odlas under standardiserade ES-cellförhållanden på gelatiniserade odlingsplattor belagda med mus embryonala fibroblaster (MEFs). Vi här förutsätter att läsaren är bekant med standardcellodlingstekniker 39-41. För att inducera differentiering, kommer ES-celler som odlas i T25 rätter separeras från MEF, och kommer att p?...

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Kombinerad DNA-RNA FISH kan vara tekniskt utmanande, eftersom förhållandena är lämpliga för DNA FISH kan vara för hård för mindre stabila RNA-molekyler. Flera tillvägagångssätt har använts för att studera både DNA och RNA i samma cell, kringgår behovet av samtidig inkubation med sökfragment som detekterar både DNA och RNA. Till exempel, i en överlagring tillvägagångssätt först RNA FISH utfördes och cellerna avbildas, och koordinaterna tas. Därefter tillämpas samma slides användes för DNA-FISH,...

Att studera celler och vävnader med hjälp av fluorescent in situ hybridisering (FISH) har, sedan den introducerades i slutet av 1970-talet 1, får forskare att studera gener, kromatin organisation och genuttryck på subcellulär nivå. DNA FISH används ofta i cytogenetik, karyotypning 2, cancerdiagnostik 3 och preimplantatorisk genetisk screening 4, och har en viktig roll i molekylär forskning 5-6 eftersom den tillåter detektion av nukleinsyror i sin nativa miljö. Single cellexpressionsanalys av RNA FISH kan upptäcka inhemska primära avskrifter och icke-kodande RNA som transkriberas från kromosomer, och erbjuder fördelar till andra tekniker som bedömer genuttryck på populationsnivå, inklusive exempelvis kvantitativ RT-PCR, genom breda uttrycksanalys eller Northern blotting . Genom att visualisera RNA-transkript med ursprung direkt från sina webbplatser ursprungs har det till exempel varitmärkt att genuttryck är stokastisk 7, och kan ibland vara allelspecifik 8. Förbättringar av tekniken har även gjort det möjligt för detektion och kvantifiering av enskilda mRNA-molekyler i celler 9-11. Den grundläggande principen för FISH består av hybridisering av nukleinsyror i cellen till en nukleinsyrasond med hjälp av starkt specifika Watson och Crick basparning. Sonden kan vara antingen direkt eller indirekt upptäckts, vilket resulterar i en signal som kan mikroskopiskt visualiseras. Inledande försök bestod av radioaktivt märkta prober, som hade nackdelar baserat på säkerhetsfrågor, begränsad rumslig upplösning och förmågan att upptäcka bara ett mål i taget 12-14. Den efterföljande utvecklingen av icke-radioaktiva etiketter, inklusive fluorokromer, haptener och enzymer, har gjort utbredd användning av fisk som en rutin molekylärbiologi teknik. FISH sonden kan antingen direkt märkt med fluorochromes genom kemisk koppling av fluorescerande molekyler till nukleinsyrasekvenser 15 och integrering av fluorescensmärkta nukleotider 16 till 19, eller sonden kan indirekt visualiseras efter integration av haptener (inklusive biotin och digoxigenin) och immunologisk detektion av haptener av hapten-specifika antikroppar konjugerade till fluorescerande reportermolekyler 20-21. Den senare metoden ger signalförstärkning genom att använda flera lager av fluorescensmärkta antikroppar, som används för att förbättra den ursprungliga signalen, och möjliggör detektion av RNA-typ som uttrycks vid låga nivåer. Genom att kombinera direkta och indirekta märkningstekniker och olika haptener kan flera mål samtidigt visualiseras inom samma cell. En av de viktigaste stegen i fisk protokoll är hybridisering av sonden till sitt mål. Även i teorin, kommer en sond specifikt binder endast till sitt mål, i praktiken, detta specificitetär inte alltid uppnås, som sönder kan binda till homologa regioner, och hybridiseringsbetingelser kommer inte alltid att vara idealisk för en viss DNA-region-eller RNA-arter. Post-hybridisering tvättar är därför av särskild betydelse, eftersom de kan öka stringensen för FISH förfarandet och kan förhindra ospecifik bindning av FISH-prober, vilket skulle resultera i en hög nivå av bakgrundsbrus. Som RNA-molekyler är enkelsträngat de lätt kan hybridiseras till en FISH sond. Däremot den dubbelsträngade DNA-molekylen behöver först ett denatureringssteg, varefter en sond kan hybridisera. Detta uppnås vanligen genom upphettning av provet, vilket resulterar i denaturering av DNA. Men under dessa svåra förhållanden, bräckligt, enkelsträngade RNA-molekyler kan förloras. Därför kräver kombinerad DNA-RNA-FISH signifikant optimering av förhållandena, och är mer tekniskt utmanande jämfört med den separata detektionen av endast RNA eller DNA. Här pre viTA detaljerat protokoll av kombinerad, samtidig DNA-RNA-FISK som har gett oss möjlighet att studera X-kromosom inaktivering (XCI) att skilja kvinnliga mus embryonala stamceller 22-24. XCI är en avgörande epigenetisk mekanism för kvinnliga foster-utveckling 25, och resulterar i heterochromatinization och därmed tysta av en av de två X-kromosomer i kvinnliga individer 26-27. Väsentligt för denna process är den icke-kodande RNA Xist 28-30, vilket regleras av RNF12 22-23 och REX1 24 proteiner. Xist uttryck blir uppregleras på framtiden inaktiva X-kromosomen (Xi) under fosterutvecklingen eller på ES-celldifferentiering in vitro , och kan spridas längs X-kromosomen och därmed locka kromatin remodeling enzymer som leder till transkription avstängning av X-kromosomen 31. Denna spridning av Xist RNA kan visualiseras genom RNA FISH såsom en beläggning av X chromonågra, som även hänvisas till som en Xist moln. Eftersom kvinnliga ES-celler kan förlora en av sina X-kromosomer på grund av genomisk instabilitet, har vi och andra anställda kombinerade DNA-RNA FISH att studera XCI, för att se till att endast karyotypiskt stabila celler bedöms i analysen av denna viktiga process 22,32 -34. Som med varje molekylärbiologi teknik har flera olika utmärkta protokoll publicerats 35-38. Här presenterar vi vår metod, från induktion av differentiering i mus ES-celler, fixering av celler, märkning av fisk sonder av Nick-translation, förbehandling av fasta celler för att möjliggöra permeabilisering och efterföljande sond upptag, hybridisering av sonden till mål, och slutligen detektering av sonden genom fluorescensmärkta antikroppar. Den häri presenterade protokollet tillåter de troende upptäcka Xist RNA och X-kromosomen inom en period på två dagar, och grunderna i denna teknik kan sannolikt anpassas till othennes system och forskningsområden.

<table border="0" cellpadding="0" cellspacing="0" width="874">
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			<td height="21" style="height:21px;width:464px;">female mouse embryonic stem cells</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">will be made available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">mouse embryonic fibroblasts</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>can be derived from E13.5 embryos, or via commercial sources e.g Millipore</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">ES cell culture medium</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>DMEM, 15% foetal calf serum, 100 U ml-1 penicillin, 100 mg ml-1 streptomycin, non-essential amino acids, 0.1 mM &#223;-mercaptoethanol, and 1000 U ml-1 LIF. Filter sterilize and store at 4&#186;C for maximal 2 weeks.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">LIF</td>
			<td style="width:124px;">Chemicon</td>
			<td style="width:119px;">ESG1107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">DMEM</td>
			<td style="width:124px;">Invitrogen</td>
			<td align="right" style="width:119px;">11960085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Fetal Calf Serum&#160;</td>
			<td style="width:124px;">Invitrogen</td>
			<td align="right" style="width:119px;">10099141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Penicillin&#8211;streptomycin (100&#215;)</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Non-essential aminoacids&#160;</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">&#223;-mercaptoethanol&#160;</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">ES cell differentiation medium</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>IMDM-Glutamax, 15% heat inactivated foetal calf serum, 100 U ml-1 penicillin, 100 mg ml-1 streptomycin, non-essential amino acids, 37.8 &#956;l/l monothioglycerol and 50 &#956;g/ml ascorbic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">IMDM-Glutamax</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">31980-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Ascorbic acid (50 mg/ml)</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">monothioglycerol</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">T25 cell culture dishes</td>
			<td style="width:124px;">Greiner Bio-one</td>
			<td align="right" style="width:119px;">690160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">6-well cell culture dishes</td>
			<td style="width:124px;">Greiner Bio-one</td>
			<td align="right" style="width:119px;">662160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">cell culture grade PBS</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Trypsin-EDTA 0.25% (v/v) trypsin/0.2% (w/v) EDTA in PBS&#160;</td>
			<td style="width:124px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">falcon tubes</td>
			<td style="width:124px;">Falcon</td>
			<td align="right" style="width:119px;">352196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">24x24 mm coverslips</td>
			<td style="width:124px;">Menzle</td>
			<td align="right" style="width:119px;">780882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">gelatin</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1890-100G</td>
			<td>prepare a 0.2% gelatin/cell culture PBS solution, and autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">paraformaldehyde</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>P6148</td>
			<td>prepare 4% PFA/PBS solutions, by dissolving PFA in PBS. Stir at 70 &#7484;C until completely dissolved and cool down to room temperature. Aliquots can be stored at -20&#7484;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">absolute 100%&#160; ethanol</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">32221-2.5L</td>
			<td>use absolute 100% ethanol and RNAse free water to make 70% and 90% ethanol solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">RNAse free water</td>
			<td style="width:124px;">Baxter</td>
			<td>TKF7114</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:464px;">sterile filter tips</td>
			<td style="width:124px;">Rainin Bio Clean</td>
			<td style="width:119px;">GP-L10F, GP-L200F, RT-1000F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">digoxigenin-labeling kit (DIG-Nick translation)</td>
			<td style="width:124px;">Roche</td>
			<td align="right" style="width:119px;">11745816910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">biotin-labeling kit (Biotin Nick translation)</td>
			<td style="width:124px;">Roche</td>
			<td align="right" style="width:119px;">11745824910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Sephadex G50</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">G5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">2 ml syringe</td>
			<td style="width:124px;">BD Plastipak</td>
			<td align="right" style="width:119px;">300013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">sterilized cotton</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">eppendorf tubes</td>
			<td style="width:124px;">Eppendorf</td>
			<td align="right" style="width:119px;">30120086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">yeast tRNA 10 mg/ml</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">15401-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Salmon Sperm DNA 10 mg/ml</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">15632-011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">mouse cot-1 DNA&#160; 1 mg/ml</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">18440-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">eppendorf bench top microcentrifuge</td>
			<td style="width:124px;">Eppendorf</td>
			<td style="width:119px;">Model 5417R&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Eppendorf Refrigerated Centrifuge</td>
			<td style="width:124px;">Eppendorf</td>
			<td style="width:119px;">Model 5810R&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">2M NaAc, pH 5.6</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">50+ hybridization solution</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>50% formamide, 2x SSC, 50 mM phosphate buffer pH 7, 10% dextran sulfate pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">formamide</td>
			<td style="width:124px;">Acros organics</td>
			<td align="right" style="width:119px;">3272350000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">dextran sulfate</td>
			<td style="width:124px;">Fluka</td>
			<td align="right" style="width:119px;">31403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">pepsin</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>P6887</td>
			<td>prepare a 0.2% solution in RNAse free water, and add 84 &#181;l 37% HCL per 100 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">HCL, 37% solution&#160;</td>
			<td style="width:124px;">Fluka</td>
			<td align="right" style="width:119px;">84422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">object glasses</td>
			<td style="width:124px;">Starfrost</td>
			<td style="width:119px;">8037/1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">20x SSC</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">AM9763</td>
			<td>dilute to 2x SSC in RNAse free water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">10 mM Phosphate buffer, pH 7</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>add 57.7 ml of 1M Na2HPO4 and 42.3 ml 1M NaH2PO4; use DEPC treated water and autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">denaturation buffer</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>70% Formamide/2xSSC/10mM phosphate buffer pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Tween-20</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">2M Tris pH 7.5 solution</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Tris-Saline-Tween washing solution (TST)</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>100 ml 10x Saline, 50 ml 2M Tris, 500 &#181;l Tween-20, add RNAse free water till 1 liter&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:464px;">10x Saline solution</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>dissolve 85 g NaCl in 1 liter RNAse free H2O, and autoclave</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:464px;">Tris-Saline-BSA (TSBSA)</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>500 &#956;l 10x Saline, 250 &#956;l 2 M Tris, 1 ml 100x BSA, 3,25 ml RNAse free H2O</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:464px;">BSA, purified, 100x&#160;</td>
			<td style="width:124px;">New England Biolabs</td>
			<td style="width:119px;">B9001S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">sheep-anti-dig antibody</td>
			<td style="width:124px;">Roche diagnostics</td>
			<td style="width:119px;"></td>
			<td>use 1:500 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit-anti-sheep antibody conjugated with FITC</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat-anti-rabbit antibody conjugated with FITC</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mouse-anti-biotin antibody</td>
			<td style="width:124px;">Roche diagnostics</td>
			<td style="width:119px;"></td>
			<td>use 1:200 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">donkey-anti-mouse antibody conjugated with Rhodamine</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat-anti-horse antibody conjugated with Rhodamine</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:464px;">Tris-Saline washing solution (TS)</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>10 ml 10x Saline, 5 ml 2 M Tris, 85 ml RNAse free H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield mounting medium with DAPI</td>
			<td style="width:124px;">Vectorlabs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nail-varnish</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">fluorescent miscroscope</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

combined dna-rna fluorescent in situ hybridization (fish) to study x chromosome inactivation in differentiated female mouse embryonic stem cells

Fluorescent in situ-hybridisering (FISH) är en molekylär teknik som gör att detektionen av nukleinsyror i celler. DNA FISH används ofta i cytogenetik och cancerdiagnostik och kan detektera avvikelser av genomet, som ofta har viktiga kliniska implikationer. RNA FISH kan användas för att detektera RNA-molekyler i celler och har gett viktiga insikter i reglering av genuttryck. Kombinera DNA och RNA FISH inom samma cell är tekniskt utmanande, eftersom förhållandena är lämpliga för DNA FISH kan vara för hårda för ömtåliga, enkelsträngade RNA-molekyler. Presenterar vi här en lätt tillämplig protokollet som gör att den kombinerade, samtidig detektion av Xist RNA och DNA kodas av X-kromosomer. Denna kombinerade DNA-RNA-FISH protokoll kan sannolikt tillämpas på andra system, där både RNA och DNA måste detekteras.

Med hjälp av ovan nämnda protokoll för kombinerad DNA-RNA-FISH har vi kunnat visualisera X-kromosom inaktivering för att skilja kvinnliga embryonala stamceller. Figur 1 visar ett representativt exempel på en DNA-RNA-FISH experiment, där vi upptäckt både Xist (vilket är synlig som en sk Xist RNA moln på den inaktiva X-kromosomen och en basal transkription peka på den aktiva X-kromosom), och en region av X-kromosomen, vilket syns som en peka signal. Observera att en av knappn?...

Watch this Scientific Journal Video about Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells at JoVE.com

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Kombinerad DNA-RNA Fluorescent<em&gt; In situ</em&gt; Hybridisering (FISH) för att studera X-kromosom inaktivering i Differentierade Kvinna Mouse embryonala stamceller

Fluorescent in situ-hybridisering (FISH) tillåter detektion av nukleinsyror i deras nativa omgivning inom celler. Vi beskriver här ett protokoll för det kombinerade, samtidig upptäckt av RNA och DNA med hjälp av FISH, som kan användas för att studera X-kromosom inaktivering i mus embryonala stamceller.

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

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Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Unit 2

Studying DNA and RNA

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Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafisk Hela Mount högupplöst Dubbel Fluorescerande In Situ Hybridisering

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Fluorescence in situ hybridization FISH Protocol in Human Sperm

Fluorescens På plats Hybridisering (FISH) Protokoll i mänskliga spermier

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

Isolering och Härledning av Mouse Embryonala Germinal celler

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Hela berget<em&gt; In situ</em&gt; Hybridisering av E8.5 till E11.5 musembryon

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

Omprogrammering Mänskliga somatiska celler in i inducerade pluripotenta stamceller (iPSCs) Använda retroviral vektor med GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

Detektering av viralt RNA genom Fluorescens<em&gt; In situ</em&gt; Hybridisering (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Analys av encelliga gentranskription av RNA-lysrör<em&gt; In Situ</em&gt; Hybridisering (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Kromosom Replikera Timing kombination med fluorescerande<em&gt; In situ</em&gt; Hybridisering

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Hel Mount RNA Lysrör<em&gt; In situ</em&gt; Hybridisering av<em&gt; Drosophila</em&gt; Embryon

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Kombinerad Immunfluorescens och DNA FISH på 3D bevarade interfaskärnorna att studera förändringar i 3D Nuclear organisation

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...