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 cellule staminali coltivate in piatti T25 saranno separati dal MEF, e saranno piastrate in medium di differenziazione. <ol><li> Rimuovere ES terreno dalla coltura di cellule ES, e lavare due volte con PBS coltura cellulare. </li><li> Aggiungere 1,5 ml di tripsina-EDTA (preriscaldata a 37 ° C), e incubare le cellule a 37 ° C per 7 minuti. Dopo 3,5 minuti, agitare delicatamente il piatto per rompere le colonie. </li><li> Inattivare la tripsina-EDTA aggiungendo 3,5 ml di terreno di differenziamento delle cellule. Cellule pipette su e giù per ottenere una soluzione singola cella, e raccoglierein una provetta da 15 ml. Spin per 5 minuti a 200 xg, e raccogliere le cellule in 10 ml di terreno di differenziamento. </li><li> Aggiungere sospensione cellulare in un piatto di coltura non-gelatinizzato, e incubare per 1 ora in un incubatore di coltura cellulare. NOTA: MEF saranno in grado di aderire al piatto di coltura non-gelatinizzato, mentre le cellule ES rimarranno in sospensione. </li><li> Nel frattempo, aggiungere vetrini sterilizzati (24 x 24 mm) a 6 pozzetti, aggiungere 0,2% di gelatina, e incubare per minimo 5 min a temperatura ambiente. </li><li> Dopo 1 ora, raccogliere il surnatante della coltura di cellule ES pre-plated, che sarà principalmente costituito da non-aderenti cellule ES. Piatto cellule ES a 6-pozzetti contenenti lamelle. NOTA: L&#39;uso ⅙ th di un piatto confluenti T25 per 6 pozzetti di una piastra da 6 pozzetti. Ciò consentirà una differenziazione confluenti e dopo 3 giorni di differenziamento. Modificare medio su base giornaliera. Per lunghi corsi a tempo differenziazione, piatto pre-plated cellule staminali embrionali in terreno di differenziamento in piatti batteriche, e incubmangiato su un agitatore cella integrata nella incubatore di coltura cellulare. Questo permetterà le cellule formano corpi embrionali, che può essere placcato al coprioggetto 1-2 giorni prima del fissaggio. </li></ol> 2. Fissazione delle cellule differenziate ES per la successiva DNA-RNA FISH Analysis <ol><li> Rimuovere il terreno di differenziamento da colture di differenziazione, e lavare due volte con coltura cellulare PBS. NOTA: Aggiungi attenzione PBS per impedire alle cellule di essere spazzati via. </li><li> Rimuovere coltura cellulare PBS e fissare cellule aggiungendo 4% paraformaldeide (PFA) / PBS e incubare per 10 min a temperatura ambiente. ATTENZIONE: PFA è tossico e può causare notevoli rischi per la salute quando viene inalato o quando è in contatto con la pelle o gli occhi. Inoltre, PFA è cancerogeno. </li><li> Rimuovere 4% PFA / PBS, e lavare coprioggetto 3x con il 70% di etanolo. NOTA: il lavaggio corretto è importante, come ogni traccia di PFA interferisce con le successive fasi di pesce. Coprioggetto possono essere memorizzati in 70% di etanolo a -20 ° C per diversi mesi prima FAnalisi ISH. </li></ol> 3. Etichettatura di sonde di DNA per gli esperimenti PESCE NOTA: Ottenere un frammento di DNA del gene di interesse (lunghezza minima&gt; 2 kb per RNA FISH, o&gt; 20 kb per FISH DNA), o un tasso alcolemico che copre il gene di interesse. Per il rilevamento di RNA, una sonda più piccola può essere sufficiente rispetto alla rilevazione del DNA, poiché molto probabilmente trascrizione darà luogo a più trascritti, che possono essere rilevati simultaneamente. Per un segnale forte sul filamento singolo di DNA copia, è necessaria una sonda più lunga, per essere in grado di rilevare un numero sufficiente di apteni incorporati. Preferibilmente, una regione genomica non trascritto è scelto per progettare la sonda di DNA. Inoltre, quando si utilizza una sonda più piccola per rilevare l&#39;RNA, si impedirà che l&#39;loci genomici da cui l&#39;RNA viene trascritto viene rilevato nell&#39;approccio FISH DNA-RNA combinato, anche se questo non può essere completamente escluso. Per rilevare topo Xist, è stata utilizzata una sonda di cDNA di 5,5 kb. Per detezione del cromosoma X, possono essere utilizzate diverse sonde BAC. Buoni risultati sono stati ottenuti con una miscela di BAC RP23-100E1 e BAC CT7-474E4, che si trovano in prossimità del locus Xist. A seconda del gene o cromosoma di interesse, più sonde differenti potrebbero essere necessari per ottenere risultati ottimali. Ogni volta che si lavora con materiali che saranno utilizzati per l&#39;RNA-FISH, utilizzare punte sterili filtri, RNasi libero H 2 O, e lavorare in un ambiente pulito. <ol><li> Prendere 1 mg di DNA e diluire in H 2 O in un volume totale di 16 microlitri. Aggiungere 4 ml di digoxigenin o soluzione di etichettatura biotina (vedi reagenti), mescolare nel vortex e incubare a 16 ° C per 90 min. NOTA: La biotina o nucleotidi digossigenina saranno ora inseriti da nick-translation. </li><li> Nel frattempo, preparare le colonne G50 per rimuovere i nucleotidi non-integrati. Prendete una siringa da 2 ml, togliere la matrice, e aggiungere il cotone sterilizzato nella siringa. Aggiungere palle G50 in soluzione to riempire la siringa, posizionare siringa in una provetta da 15 ml e centrifugare a 642 xg per 1 min. NOTA: Circa l&#39;80% della siringa dovrebbe ora essere riempito con G50. Ripetere quando meno G50 è presente. </li><li> Dopo 90 minuti, aggiungere 40 ml di H 2 O alla miscela di reazione nick-translation, e aggiungere la reazione alla colonna G50. Aggiungere un tubo vuoto nella colonna, e centrifugare a 642 xg per 2 minuti. Raccogliere il flusso attraverso un tubo di reazione. </li><li> Misurare il volume del flusso attraverso con una pipetta e aggiungere H 2 O per raggiungere un volume totale di 200 microlitri. Precipitare il flusso attraverso l&#39;aggiunta di 13,3 ml di DNA di sperma di salmone (10 mg / ml), 13,3 microlitri tRNA di lievito (10 mg / ml), 26,7 microlitri topo Cot-1 DNA (1 mg / ml) e 28 microlitri 2 M NaAc, pH 5.6. Mescolare nel vortex, e aggiungere 533 ml di ghiaccio freddo al 100% di etanolo. Agitare la provetta e centrifugare a 16.200 xg per 30 min a 4 ° C. </li><li> Rimuovere il surnatante, e lavare pellet due volte con etanolo al 70%. Centrifugare a 16.200 xg per 5 min a4 ° C, e asciugare il pellet. Aggiungere 50 ml di soluzione al 50 + ibridazione, e incubare a 37 ° C per 30 min per facilitare dissoluzione. NOTA: etichetta sonda può essere conservato per diversi anni a -20 ° C. ATTENZIONE: 50 + soluzione di ibridazione contenente formammide, che è tossico, può irritare la pelle, gli occhi e l&#39;apparato respiratorio, ed è anche un teratogeno. </li></ol> 4. Permeabilization, pre-trattamento e ibridazione di cellule fissate per Combined DNA-RNA FISH <ol><li> Reidratare cellule fissate su vetrini, rimuovendo il 70% di etanolo e l&#39;aggiunta della coltura cellulare PBS alle piastre a 6 pozzetti. Incubare per 5 min. Ripetere in totale tre volte. </li><li> Rimuovere coltura cellulare PBS e aggiungere 0,2% pepsina alle piastre da 6 pozzetti, per permeabilize le cellule. Incubare le piastre a 6 pozzetti in un bagno d&#39;acqua a 37 ° C per 4 min. Rimuovere pepsina e inattivare con RNAsi libera H 2 O. </li><li> Cellule post-fix di incubazione con il 4% PFA / PBS per 5 minuti a temperatura ambiente. Dopo fixatisu, lavare due volte con PBS coltura cellulare per 5 min. </li><li> Disidratare incubando le cellule con etanolo al 70% per 3 min, 90% di etanolo per 3 min e 100% etanolo per 3 min. Trasferimento coprioggetto su piastre da 6 pozzetti e coprioggetto secco aria per diversi minuti. </li><li> Età cellule di incubazione vetrini a 65 ° C per 1 ora su un piatto caldo. </li><li> Pre-ibridare sonda FISH con il mouse Cot-1 DNA sommando per ogni vetrino da analizzare, 25 ml di soluzione di 50 + ibridazione, 0,5 microlitri del mouse Cot-1 DNA, 1 ml di sonda Xist biotina marcata e 1 ml di digossigenina sonda BAC marcata rilevare il cromosoma X. Mescolare nel vortex e denaturare a 99 ° C per 5 min. Incubare immediatamente a 37 ° C per 45 minuti, per consentire Cot-1 DNA per ibridare ripetere sequenze presenti nelle sonde. </li><li> Dopo l&#39;invecchiamento, individuare 50 microlitri di tampone di denaturazione (comprensivi di 70% formamide/2x tampone fosfato SSC/10 mM) su un vetrino e coprioggetto mettere in cima con cellule rivolto towards buffer denaturazione. Incubare a 65 ° C per 2 min. </li><li> Aggiungi coprioggetto torna alla 6 pozzetti e cellule post-fix incubando in ghiaccio freddo etanolo al 70% per 5 min. </li><li> Disidratare cellule da successiva incubazione in etanolo al 70% per 3 min, 90% di etanolo per 3 min e 100% etanolo per 3 min. Rimuovere il coprioggetto da 6 pozzetti e lasciare asciugare per alcuni minuti. </li><li> Spot sonda pre-ibridato su un vetrino, e incubare coprioggetto sulla parte superiore. Posizionare i vetrini in una camera umidificata riempita con 50 ml 50% formamide/2x SSC, e incubare una notte a 37 ° C. </li></ol> 5. Lavaggi post-ibridazione e anticorpi Mediated Rilevamento di sonda <ol><li> Fill 6 pozzetti con 2x SSC, e pre-riscaldare fino a 42 ° C. Aggiungere coprioggetto dopo incubazione notturna, e incubare a 42 ° C per 5 min. </li><li> Rimuovere 2x SSC, e incubare coprioggetto con il 50% formamide/2x SSC per 10 minuti a 42 ° C. Ripetere in totale 3 volte (30 min di lavaggio del tutto). </li&gt; <li> Rimuovere 50% formamide/2x SSC, e lavare i vetrini con Tris-salino-Tween (TST) da 2 x 5 min a temperatura ambiente. </li><li> Spot 50 ml Tris-salino-BSA (TSBSA) per vetrino sui nuovi vetrini e incubare vetrini a testa in giù per bloccare durante 30 minuti a temperatura ambiente in una camera oscura TST-umidificata. </li><li> Trasferimento coprioggetto torna a 6 pozzetti e lavare per 2 x 5 minuti con TST. </li><li> Spot 50 ml di anticorpi di pecora anti-dig (1:500 in TSBSA) per vetrino sui nuovi vetrini e incubare coprioggetto upside-down per la prima fase di incubazione per 30 minuti a temperatura ambiente in una camera oscura TST-umidificata. </li><li> Trasferimento coprioggetto torna a 6 pozzetti e lavare per 2 x 5 minuti con TST. </li><li> Spot 50 ml di coniglio anti-pecora anticorpo coniugato con FITC (1:250 in TSBSA) per vetrino sui nuovi vetrini e incubare vetrini a testa in giù per la seconda fase di incubazione per 30 minuti a temperatura ambiente in una camera oscura TST-umidificata . </li><li> Transfer coprioggetto torna a 6 pozzetti e lavare per 2 x 5 minuti con TST. </li><li> Spot 50 ml di anticorpi di capra anti-coniglio coniugato con FITC (1:250 in TSBSA) per vetrino sui nuovi vetrini e incubare vetrini a testa in giù per la terza fase di incubazione per 30 minuti a temperatura ambiente in una camera oscura TST-umidificata . </li><li> Trasferimento coprioggetto torna a 6 pozzetti e lavare per 2 x 5 minuti con TST. </li><li> Spot 50 ml di topo anti-biotina anticorpi (1:200 in TSBSA) per vetrino sui nuovi vetrini e incubare lamelle a testa in giù per la quarta fase di incubazione durante 30 minuti a temperatura ambiente in una camera oscura TST-umidificata. </li><li> Trasferimento coprioggetto torna a 6 pozzetti e lavare per 2 x 5 minuti con TST. </li><li> Spot 50 ml di anticorpi asino-anti-topo coniugati con rodamina (1:250 in TSBSA) per vetrino sui nuovi vetrini e incubare lamelle a testa in giù per la quinta fase di incubazione durante 30 minuti a temperatura ambiente in un chamb scuro TST-umidificataER. </li><li> Trasferimento coprioggetto torna a 6 pozzetti e lavare per 2 x 5 minuti con TST. </li><li> Spot 50 ml di anticorpi di capra anti-cavallo coniugata con Rhodamine (1:250 in TSBSA) per vetrino sui nuovi vetrini e incubare vetrini a testa in giù per la sesta fase di incubazione durante 30 minuti a temperatura ambiente in una camera oscura TST-umidificata . NOTA: l&#39;anticorpo di capra anti-cavallo riconoscerà l&#39;anticorpo asino-anti-topo; quando a disposizione del ricercatore, un anticorpo di capra anti-donkey funzionerà pure. </li><li> Trasferimento coprioggetto torna a 6 pozzetti e lavare per 2 x 5 minuti con TST. Lavare ulteriore 5 min con Tris-salino (TS). </li><li> Disidratare cellule da successiva incubazione in etanolo al 70% per 3 min, 90% di etanolo per 3 min e 100% etanolo per 3 min. Rimuovere il coprioggetto da 6 pozzetti e lasciare asciugare per alcuni minuti. </li><li> Identifica una goccia di mezzo di montaggio con DAPI (vedi reagenti) su un vetrino e aggiungere coprioggetto a testa in giù in cima. Sigillare i vetrini con smalto evalutare i risultati FISH mediante microscopia a fluorescenza (Figura 1). </li></ol>

<ol>
	<li>Rudkin, G. T., Stollar, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+detection+of+DNA-RNA+hybrids+in+situ+by+indirect+immunofluorescence.">High resolution detection of DNA-RNA hybrids in situ by indirect immunofluorescence.</a> Nature. 265, 472-473 (1977).</li><li>Imataka, G., Arisaka, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+analysis+using+spectral+karyotyping+(SKY).">Chromosome analysis using spectral karyotyping (SKY).</a> Cell Biochem Biophys. 62, 13-17 (2012).</li><li>Ross, F. 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=Report+from+the+European+Myeloma+Network+on+interphase+FISH+in+multiple+myeloma+and+related+disorders.">Report from the European Myeloma Network on interphase FISH in multiple myeloma and related disorders.</a> Haematologica. 97, 1272-1277 (2012).</li><li>Mastenbroek, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preimplantation+genetic+screening:+a+systematic+review+and+meta-analysis+of+RCTs.">Preimplantation genetic screening: a systematic review and meta-analysis of RCTs.</a> Hum Reprod Update. 17, 454-466 (2011).</li><li>Martins-Taylor, K., Xu, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+Genomic+stability+of+human+induced+pluripotent+stem+cells.">Concise review: Genomic stability of human induced pluripotent stem cells.</a> Stem Cells. 30, 22-27 (2012).</li><li>Rens, 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=Resolution+and+evolution+of+the+duck-billed+platypus+karyotype+with+an+X1Y1X2Y2X3Y3X4Y4X5Y5+male+sex+chromosome+constitution.">Resolution and evolution of the duck-billed platypus karyotype with an X1Y1X2Y2X3Y3X4Y4X5Y5 male sex chromosome constitution.</a> Proc Natl Acad Sci U S A. 101, 16257-16261 (2004).</li><li>Munsky, 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=Using+gene+expression+noise+to+understand+gene+regulation.">Using gene expression noise to understand gene regulation.</a> Science. 336, 183-187 (2012).</li><li>Miyanari, Y., Torres-Padilla, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+ground-state+pluripotency+by+allelic+regulation+of+Nanog.">Control of ground-state pluripotency by allelic regulation of Nanog.</a> Nature. 483, 470-473 (2012).</li><li>Kwon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+fluorescence+in+situ+hybridization:+quantitative+imaging+of+single+RNA+molecules.">Single-molecule fluorescence in situ hybridization: quantitative imaging of single RNA molecules.</a> BMB Rep. 46, 65-72 (2013).</li><li>Ji, N., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+molecule+fluorescent+in+situ+hybridization+(smFISH)+of+C.+elegans+worms+and+embryos.">Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos.</a> WormBook. , 1-16 (2012).</li><li>Femino, 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=Visualization+of+single+RNA+transcripts+in+situ.">Visualization of single RNA transcripts in situ.</a> Science. 280, 585-590 (1998).</li><li>Buongiorno-Nardelli, M., Amaldi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoradiographic+detection+of+molecular+hybrids+between+RNA+and+DNA+in+tissue+sections.">Autoradiographic detection of molecular hybrids between RNA and DNA in tissue sections.</a> Nature. 225, 946-948 (1970).</li><li>Gall, J. G., Pardue, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+detection+of+RNA-DNA+hybrid+molecules+in+cytological+preparations.">Formation and detection of RNA-DNA hybrid molecules in cytological preparations.</a> Proc Natl Acad Sci U S A. 63, 378-383 (1969).</li><li>John, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-DNA+hybrids+at+the+cytological+level.">RNA-DNA hybrids at the cytological level.</a> Nature. 223, 582-587 (1969).</li><li>Bauman, 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=A+new+method+for+fluorescence+microscopical+localization+of+specific+DNA+sequences+by+in+situ+hybridization+of+fluorochromelabelled+RNA.">A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochromelabelled RNA.</a> Exp Cell Res. 128, 485-490 (1980).</li><li>Wiegant, 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=In+situ+hybridization+with+fluoresceinated+DNA.">In situ hybridization with fluoresceinated DNA.</a> Nucleic Acids Res. 19, 3237-3241 (1991).</li><li>Langer, P. R., 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=Enzymatic+synthesis+of+biotin-labeled+polynucleotides:+novel+nucleic+acid+affinity+probes.">Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes.</a> Proc Natl Acad Sci U S A. 78, 6633-6637 (1981).</li><li>Manuelidis, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+mapping+of+satellite+DNA+using+biotin-labeled+DNA+probes.">High-resolution mapping of satellite DNA using biotin-labeled DNA probes.</a> J Cell Biol. 95, 619-625 (1982).</li><li>Kislauskis, E. H., 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=Isoform-specific+3'-untranslated+sequences+sort+alpha-cardiac+and+beta-cytoplasmic+actin+messenger+RNAs+to+different+cytoplasmic+compartments.">Isoform-specific 3'-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments.</a> J Cell Biol. 123, 165-172 (1993).</li><li>Singer, R. H., Ward, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+gene+expression+visualized+in+chicken+muscle+tissue+culture+by+using+in+situ+hybridization+with+a+biotinated+nucleotide+analog.">Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog.</a> Proc Natl Acad Sci U S A. 79, 7331-7335 (1982).</li><li>Corput, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+using+horseradish+peroxidase-labeled+oligodeoxynucleotides+and+tyramide+signal+amplification+for+sensitive+DNA+and+mRNA+detection.">Fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification for sensitive DNA and mRNA detection.</a> Histochem Cell Biol. 110, 431-437 (1998).</li><li>Jonkers, I., 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=RNF12+is+an+X-Encoded+dose-dependent+activator+of+X+chromosome+inactivation.">RNF12 is an X-Encoded dose-dependent activator of X chromosome inactivation.</a> Cell. 139, 999-101 (2009).</li><li>Barakat, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNF12+activates+Xist+and+is+essential+for+X+chromosome+inactivation.">RNF12 activates Xist and is essential for X chromosome inactivation.</a> PLoS Genet. 7, (2011).</li><li>Gontan, C., 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=RNF12+initiates+X-chromosome+inactivation+by+targeting+REX1+for+degradation.">RNF12 initiates X-chromosome inactivation by targeting REX1 for degradation.</a> Nature. 485, 386-390 (2012).</li><li>Lyon, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+action+in+the+X-chromosome+of+the+mouse+(Mus+musculus+L).">Gene action in the X-chromosome of the mouse (Mus musculus L).</a> Nature. 190, 372-373 (1961).</li><li>Barakat, T. S., Gribnau, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=chromosome+inactivation+in+the+cycle+of+life.">chromosome inactivation in the cycle of life.</a> Development. 139, 2085-2089 (2012).</li><li>Augui, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+X-chromosome+inactivation+by+the+X-inactivation+centre.">Regulation of X-chromosome inactivation by the X-inactivation centre.</a> Nat Rev Genet. 12, 429-442 (2011).</li><li>Brown, C. 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+gene+from+the+region+of+the+human+X+inactivation+centre+is+expressed+exclusively+from+the+inactive+X+chromosome.">A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome.</a> Nature. 349, 38-44 (1991).</li><li>Brockdorff, N., 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=Conservation+of+position+and+exclusive+expression+of+mouse+Xist+from+the+inactive+X+chromosome.">Conservation of position and exclusive expression of mouse Xist from the inactive X chromosome.</a> Nature. 351, 329-331 (1991).</li><li>Borsani, 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=Characterization+of+a+murine+gene+expressed+from+the+inactive+X+chromosome.">Characterization of a murine gene expressed from the inactive X chromosome.</a> Nature. 351, 325-329 (1991).</li><li>Barakat, T. S., Gribnau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X+chromosome+inactivation+and+embryonic+stem+cells.">X chromosome inactivation and embryonic stem cells.</a> Adv Exp Med Biol. 695, 132-154 (2010).</li><li>Chaumeil, 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+novel+role+for+Xist+RNA+in+the+formation+of+a+repressive+nuclear+compartment+into+which+genes+are+recruited+when+silenced.">A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced.</a> Genes Dev. 20, 2223-2237 (2006).</li><li>Okamoto, I., 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=Eutherian+mammals+use+diverse+strategies+to+initiate+X-chromosome+inactivation+during+development.">Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development.</a> Nature. 472, 370-374 (2011).</li><li>Gribnau, 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=X+chromosome+choice+occurs+independently+of+asynchronous+replication+timing.">X chromosome choice occurs independently of asynchronous replication timing.</a> J Cell Biol. 168, 365-373 (2005).</li><li>Namekawa, S. H., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+nascent+RNA,+single-copy+DNA+and+protein+localization+by+immunoFISH+in+mouse+germ+cells+and+preimplantation+embryos.">Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos.</a> Nat Protoc. 6, 270-284 (2011).</li><li>Van Tine, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+in+situ+detection+of+RNA,+DNA,+and+protein+using+tyramide-coupled+immunofluorescence.">Simultaneous in situ detection of RNA, DNA, and protein using tyramide-coupled immunofluorescence.</a> Methods Mol Biol. 292, 215-230 (2005).</li><li>Pollex, T., 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=Live-Cell+Imaging+Combined+with+Immunofluorescence,+RNA,+or+DNA+FISH+to+Study+the+Nuclear+Dynamics+and+Expression+of+the+X-Inactivation+Center.">Live-Cell Imaging Combined with Immunofluorescence, RNA, or DNA FISH to Study the Nuclear Dynamics and Expression of the X-Inactivation Center.</a> Methods Mol Biol. 1042, 13-31 (2013).</li><li>Chaumeil, 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=Combined+immunofluorescence,+RNA+fluorescent+in+situ+hybridization,+and+DNA+fluorescent+in+situ+hybridization+to+study+chromatin+changes,+transcriptional+activity,+nuclear+organization,+and+X-chromosome+inactivation.">Combined immunofluorescence, RNA fluorescent in situ hybridization, and DNA fluorescent in situ hybridization to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation.</a> Methods Mol Biol. 463, 297-308 (2008).</li><li>Lin, S., Talbot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+culturing+mouse+and+human+embryonic+stem+cells.">Methods for culturing mouse and human embryonic stem cells.</a> Methods Mol Biol. 690, 31-56 (2011).</li><li>Meissner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+manipulation+of+murine+embryonic+stem+cells.">Derivation and manipulation of murine embryonic stem cells.</a> Methods Mol Biol. 482, 3-19 (2009).</li><li>Nichols, J., Ying, Q. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+propagation+of+embryonic+stem+cells+in+serum-+and+feeder-free+culture.">Derivation and propagation of embryonic stem cells in serum- and feeder-free culture.</a> Methods Mol Biol. 329, 91-98 (2006).</li></ol>

Gli autori non hanno nulla da rivelare.

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

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.

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

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

combined-dna-rna-fluorescent-situ-hybridization-fish-to-study-x

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

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

SCIENTISTS IN ACTION

27.5K Views.  Erasmus MC - University Medical Center.  Ibridazione in situ fluorescente (FISH) permette la rilevazione di acidi nucleici nel loro ambiente nativo all'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. 

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

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 ...

Ricerca

Biologia

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

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

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

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 is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

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

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old 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 ...

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

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

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)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

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

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

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

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

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

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

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

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

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 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...