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. Induzione del differenziamento in cellule staminali embrionali femminile per indurre inattivazione del cromosoma X NOTA: le cellule staminali embrionali di topo femmina (disponibili su richiesta) sono cresciuti in condizioni di celle standard ES su piastre di coltura gelatinizzati rivestiti con fibroblasti embrionali di topo (MEF). Siamo qui per scontato che il lettore abbia familiarità con le tecniche standard di coltura cellulare 39-41. Per indurre il differenziamento, le cell...

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Combinato DNA-RNA pesce può essere tecnicamente impegnativo, in quanto le condizioni adatte per FISH DNA può essere troppo duro per le molecole di RNA meno stabili. Diversi approcci sono stati applicati per studiare sia DNA e RNA nella stessa cella, aggirando la necessità di incubazione simultanea con sonde rilevando sia il DNA e RNA. Ad esempio, in un approccio di sovrapposizione, viene eseguita prima FISH RNA, e le cellule vengono esposte, e le coordinate sono prese. Successivamente, gli stessi vetrini verranno usa...

Studiando cellule e tessuti mediante ibridazione in situ fluorescente (FISH) ha, sin dalla sua introduzione nel tardo 1970 1, ha permesso ai ricercatori di studiare i geni, l&#39;organizzazione della cromatina e l&#39;espressione genica a livello subcellulare. FISH DNA è spesso usato in citogenetica, cariotipo 2, diagnostica oncologica 3 e pre-impianto screening genetico 4, ed ha un ruolo importante nella ricerca molecolare 5-6 in quanto permette la rilevazione di acidi nucleici nel loro ambiente nativo. Singolo analisi di espressione cella RNA FISH in grado di rilevare trascritti primari autoctone ed RNA non codificanti essere trascritto da cromosomi, e offre vantaggi ad altre tecniche che valutano l&#39;espressione genica a livello di popolazione, tra cui ad esempio RT-PCR quantitativa, genoma vasta analisi di espressione o Northern blotting . Visualizzando trascritti di RNA provenienti direttamente dai loro luoghi di origine, è stato per esempionotato che l&#39;espressione genica è stocastico 7, e può a volte essere allele specifico 8. Miglioramenti nella tecnica hanno anche permesso l&#39;individuazione e la quantificazione di molecole di mRNA singoli all&#39;interno delle cellule 9-11. Il principio di base della FISH consiste di ibridazione di acidi nucleici all&#39;interno della cella a una sonda di acido nucleico mediante altamente specifico Watson e Crick base-accoppiamento. La sonda può essere direttamente o indirettamente rilevato, risultando in un segnale che può essere visualizzata microscopicamente. I tentativi iniziali consistevano di sonde marcati radioattivamente, che aveva inconvenienti basato su questioni di sicurezza, la risoluzione spaziale limitata e la capacità di rilevare solo un obiettivo alla volta 12-14. Il successivo sviluppo di marchi non radioattivi, tra cui fluorocromi, apteni, ed enzimi, ha permesso l&#39;uso diffuso di pesce come una tecnica di biologia molecolare di routine. La sonda FISH può o essere etichettato direttamente con fluorochRomes collegando chimica delle molecole fluorescenti di acido nucleico sequenze 15 e l&#39;integrazione dei nucleotidi fluorescente 16-19, o la sonda può essere indirettamente visualizzate dopo integrazione di apteni (compresi biotina e digossigenina) e rivelazione immunologica di apteni da anticorpi specifici coniugati con aptene reporter fluorescente molecole 20-21. Quest&#39;ultimo approccio permette l&#39;amplificazione del segnale utilizzando diversi strati di fluorescente anticorpi che vengono utilizzati per migliorare il segnale originale, e consente il rilevamento di specie di RNA che sono espressi a livelli bassi. Combinando tecniche di etichettatura diretti e indiretti e vari apteni, diversi obiettivi possono essere contemporaneamente visualizzati all&#39;interno della stessa cella. Uno dei passi più importanti nei protocolli di pesce è l&#39;ibridazione della sonda al suo obiettivo. Sebbene in teoria, una sonda verrà specificatamente legarsi solo al suo bersaglio, in pratica, questa specificitànon è sempre raggiunto, come sonde possono legarsi a regioni omologhe, e condizioni di ibridazione non sempre ideale per una certa regione di DNA o RNA specie. Lavaggi post-ibridazione sono quindi di particolare importanza, in quanto possono aumentare la severità della procedura FISH, e possono impedire il legame non specifico di sonde FISH, che porterebbe a un elevato livello di rumore di fondo. Come molecole di RNA sono a singolo filamento che possano essere facilmente ibridate ad una sonda FISH. Al contrario, la molecola di DNA a doppio filamento deve prima una fase di denaturazione, dopo che una sonda può ibridare. Ciò viene ottenuto mediante riscaldamento del campione, che provoca la denaturazione del DNA. Tuttavia, in queste condizioni difficili, fragili molecole di RNA a singolo filamento potrebbero essere persi. Pertanto, DNA-RNA combinato FISH richiede significativa ottimizzazione delle condizioni, ed è tecnicamente più difficile rispetto alla rilevazione separata di soli RNA o DNA. Qui Presenta protocollo dettagliato della produzione combinata e simultanea FISH DNA-RNA che ci ha permesso di studiare inattivazione del cromosoma X (XCI) nella differenziazione delle cellule staminali embrionali di topo femmina 22-24. XCI è un meccanismo epigenetico cruciale per lo sviluppo embrionale femmina 25, e si traduce in heterochromatinization e quindi silenziamento di uno dei due cromosomi X in individui femmina 26-27. Essenziale per questo processo è l&#39;RNA non codificante Xist 28-30, che è regolata dalle RNF12 22-23 e Rex1 24 proteine. Espressione Xist diventa sovraregolati sul futuro cromosoma X inattivo (Xi) durante lo sviluppo embrionale o sulla differenziazione delle cellule ES in vitro , e può diffondersi lungo il cromosoma X e, quindi, ottenere il rimodellamento della cromatina enzimi che provocano l&#39;arresto della trascrizione del cromosoma X 31. Questa diffusione di Xist RNA possono essere visualizzati da RNA FISH come rivestimento di X cromoalcuni, che è indicato anche come una nube Xist. Dal momento che le cellule staminali femminili possono perdere uno dei loro cromosomi X a causa di instabilità genomica, noi ed altri abbiamo impiegato combinato DNA-RNA FISH a studiare XCI, fare in modo che le cellule solo karyotypically stabili sono valutati nell&#39;analisi di questo importante processo 22,32 -34. Come per ogni tecnica di biologia molecolare, diversi protocolli diversi eccellenti sono stati pubblicati 35-38. Presentiamo qui il nostro metodo, partendo dalla induzione del differenziamento in cellule ES di topo, fissazione delle cellule, etichettatura di sonde FISH di Nick-traduzione, pretrattamento di cellule fissate per consentire permeabilizzazione e conseguente assorbimento sonda, l&#39;ibridazione della sonda al bersaglio, e infine il rilevamento della sonda fluorescente anticorpi. Il protocollo qui presentato permette la rilevazione fedeli di Xist RNA e il cromosoma X entro due giorni, e le basi di questa tecnica può probabilmente essere adattata per oti suoi sistemi e settori di ricerca.

<table border="0" cellpadding="0" cellspacing="0" width="874">
	<tbody>
		<tr height="21">
			<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

Ibridazione in situ fluorescente (FISH) è una tecnica molecolare che consente il rilevamento di acidi nucleici nelle cellule. FISH DNA è spesso usato in citogenetica e diagnostica del cancro, e può rilevare aberrazioni del genoma, che spesso ha importanti implicazioni cliniche. RNA FISH può essere utilizzato per rilevare molecole di RNA in cellule e ha fornito spunti importanti nella regolazione dell&#39;espressione genica. La combinazione di DNA e RNA FISH all&#39;interno della stessa cellula è tecnicamente impegnativo, in quanto le condizioni adatte per FISH DNA potrebbe essere troppo duro per i fragili, molecole di RNA a singolo filamento. Riportiamo un protocollo facilmente applicabile, che consente, rilevazione simultanea combinata di Xist RNA e DNA codificato dai cromosomi X. Questo protocollo FISH DNA-RNA combinato può probabilmente essere applicata ad altri sistemi in cui sia RNA e DNA devono essere rilevata.

Utilizzando il protocollo di cui sopra per il combinato DNA-RNA FISH, siamo stati in grado di visualizzare inattivazione del cromosoma X nella differenziazione delle cellule staminali embrionali femminili. Figura 1 mostra un esempio rappresentativo di un esperimento FISH DNA-RNA, dove abbiamo rilevato sia Xist (che è visibile come un cosiddetto Xist RNA nube sul cromosoma X inattivo e un puntino trascrizione basale sul cromosoma X attivo), e una regione del cromosoma X, che è visibil...

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

DNA-RNA combinato fluorescente<em&gt; In situ</em&gt; Ibridazione (FISH) per studiare inattivazione del cromosoma X nelle cellule staminali embrionali di topo differenziata Femminile

Ibridazione in situ fluorescente (FISH) permette la rilevazione di acidi nucleici nel loro ambiente nativo all&#39;interno delle cellule. Noi descriviamo qui un protocollo per la, rilevazione simultanea combinata di RNA e DNA mediante FISH, che può essere usato per studiare inattivazione del cromosoma X in cellule staminali embrionali di topo.

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

Zebrafish intero monte ad alta risoluzione doppia fluorescente In Situ Ibridazione

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

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Biologia

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Fluorescence in situ hybridization FISH Protocol in Human Sperm

Fluorescenza In situ</emIbridazione> (FISH) Protocollo di sperma umano

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

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

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

Tutto il monte In Situ</emIbridazione> di E8.5 a E11.5 embrioni di topo

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

Riprogrammare cellule somatiche umane in cellule staminali pluripotenti indotte (iPSCs) Utilizzo vettore retrovirale con 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

Rilevamento di RNA virale per fluorescenza<em&gt; In situ</em&gt; Hybridization (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

Analisi di Single-cell trascrizione del gene per l&#39;RNA fluorescente<em&gt; In Situ</em&gt; Ibridazione (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

Cromosoma Timing Replica combinazione con fluorescente<em&gt; In situ</em&gt; Ibridazione

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

Tutta la Monte RNA fluorescente<em&gt; In situ</em&gt; L&#39;ibridazione di<em&gt; Drosophila</em&gt; Embrioni

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

Immunofluorescenza combinato e FISH DNA 3D conservato nuclei in interfase di studiare i cambiamenti in 3D Organizzazione nucleare

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