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. Indução de Diferenciação no feminino as células estaminais embrionárias para induzir inativação do cromossomo X NOTA: células-tronco embrionárias de mulher (disponíveis mediante solicitação) são cultivadas em condições normais de células ES em placas de cultura gelatinizadas revestidas com fibroblastos de rato embrionárias (MEFs). Nós aqui assumimos que o leitor está familiarizado com as técnicas de cultura de células normais 39-41. Para induzir a diferenciação de células ES cultivadas em pratos T25 será separada das MEFs, e vai ser plaqueadas em meio de diferenciação. <ol><li> Remova o meio ES a partir da cultura de células ES, e lavar duas vezes com PBS de cultura de células. </li><li> Adicionar 1,5 ml de tripsina-EDTA (pré-aquecido a 37 ° C), e incuba-se as células a 37 ° C durante 7 min. Depois de 3,5 min, agitar o prato delicadamente para quebrar as colônias. </li><li> Inactivar a tripsina-EDTA, adicionando 3,5 ml de meio de diferenciação das células. Células de pipeta cima e para baixo para obter uma solução única célula, e recolherem um tubo de 15 ml. Centrifugação durante 5 minutos a 200 xg, e recolher as células em 10 ml de meio de diferenciação. </li><li> Adicionar a suspensão de células para um prato de cultura não gelatinizado, e incubar durante 1 hora numa incubadora de cultura de células. NOTA: MEFs serão capazes de aderir à placa de cultura não gelatinizado, enquanto que as células ES irão permanecer em suspensão. </li><li> No entanto, adicionar lamelas de vidro esterilizado (24 x 24 mm) em placas de 6 poços, adicionar 0,2% de gelatina, e incubar durante um mínimo de 5 min à temperatura ambiente. </li><li> Depois de 1 hora, recolher o sobrenadante da cultura de células ES de pré-banhado, que consistem principalmente de não-aderentes células-tronco embrionárias. Placa células-tronco embrionárias para placas de 6 poços contendo lamínulas. NOTA: Use ⅙ º de um prato confluente T25 por 6 poços de uma placa de 6 poços. Isto irá permitir uma diferenciação bem confluentes depois de 3 dias de diferenciação. Mudança de meio, numa base diária. Para cursos mais longos de tempo diferenciação, placa pré-banhado células-tronco embrionárias em meio de diferenciação em pratos bacterianas e incubcomi num agitador célula integrada na incubadora de cultura de células. Isto irá permitir que as células de modo a formar corpos embrióides, que podem ser plaqueadas a lamelas 1-2 dias antes da fixação. </li></ol> 2. Fixação de células diferenciadas ES para posterior análise FISH DNA-RNA <ol><li> Remova o meio de diferenciação de culturas de diferenciação, e lavar duas vezes com cultura de células PBS. NOTA: Adicionar PBS com cuidado para evitar que as células serem lavados. </li><li> Retirar cultura celular PBS e fixar as células pela adição de 4% de paraformaldeído (PFA) / PBS, e incubar durante 10 min à temperatura ambiente. CUIDADO: PFA é tóxico e pode causar risco significativo para a saúde ao ser inalado ou em contato com a pele ou olhos. Além disso, PFA é cancerígeno. </li><li> Retirar 4% PFA / PBS, e lavar 3x lamelas com etanol a 70%. NOTA: lavagem correta é importante, pois qualquer traço de PFA irá interferir com passos PEIXES subseqüentes. As lamelas podem ser armazenados em etanol a 70% a -20 ° C durante vários meses antes de FAnálise ISH. </li></ol> 3. Rotulagem de DNA Sondas para Experimentos PEIXES NOTA: obter um fragmento de DNA do gene de interesse (comprimento mínimo&gt; 2 kb de RNA FISH, ou&gt; 20 kb de DNA FISH) ou a BAC cobrindo o gene de interesse. Para a detecção de ARN, uma sonda de menor pode ser suficiente em comparação com a detecção de ADN, uma vez que a transcrição provavelmente irá resultar em múltiplos transcritos, que podem ser detectadas simultaneamente. Para um sinal forte na única cadeia de ADN exemplar, uma sonda não é necessário, para ser capaz de detectar um número suficiente de haptenos incorporadas. De preferência, uma região genómica não transcrita é escolhido para a concepção da sonda de ADN. Além disso, quando se usa uma sonda para detectar o mais pequeno ARN, que vai ser impedido que o locus genómico a partir do qual o ARN é transcrito está a ser detectado na abordagem PEIXES DNA-RNA, combinados, apesar de este não pode ser totalmente excluído. Para detectar rato Xist, foi utilizada uma sonda de cDNA de 5,5 kb. Para detexão do cromossoma X, várias sondas BAC pode ser usado. Bons resultados têm sido obtidos com uma mistura de BAC RP23-100E1 e BAC CT7-474E4, que estão localizados em estreita proximidade com o locus Xist. Dependendo do gene ou cromossomo de interesse, várias sondas diferentes podem ser necessários para obter os melhores resultados. Sempre que trabalhar com materiais que serão utilizados para o RNA-FISH, utilize pontas estéreis filtro, RNAse livre H 2 O, e trabalhar em um ambiente limpo. <ol><li> Tomar 1 ug de ADN e dilua em H 2 O para um volume total de 16 ul. Adicione 4 mL de solução de etiquetagem digoxigenina ou biotina (ver reagentes), misture por vórtex e incubar a 16 ° C por 90 min. NOTA: A biotina ou nucleotídeos marcados com digoxigenina serão agora incorporadas por nick-translation. </li><li> Enquanto isso, prepare colunas G50 para remover nucleotídeos não-integrados. Tomar uma seringa de 2 ml, remover o batedor, e adicionar de algodão esterilizado para dentro da seringa. Adicionar bolas G50 em solução to preenchimento da seringa, seringa colocar num tubo de 15 ml e centrifugar a 642 xg durante 1 min. NOTA: Cerca de 80% da seringa deve agora ser preenchido com G50. Repita quando menos G50 está presente. </li><li> Após 90 min, adicionar 40 mL de H2O à mistura de reacção de tradução de nick-, e adicionar a reacção para a coluna G50. Adicionar um tubo vazio na coluna, e centrifugar a 642 xg durante 2 min. Recolhe-se o fluxo através de um tubo de reacção. </li><li> Medir o volume do fluxo através de uma pipeta, e adicionar H 2 O para atingir um volume total de 200 ul. Precipitar o fluxo através de adição de 13,3 ul de ADN de esperma de salmão (10 mg / mL), 13,3 mL de tRNA de levedura (10 mg / ml), 26,7 ul de rato Cot-1 DNA (1 mg / ml) e 28 ul de 2 M de NaAc, pH 5.6. Misture em vortex, e adicionar 533 mL de gelo frio etanol 100%. Agitar o tubo, e centrifuga-se a 16.200 xg durante 30 min a 4 ° C. </li><li> Retirar o sobrenadante, e lave o peletizado duas vezes com etanol a 70%. Centrifugar a 16.200 xg durante 5 min a4 ° C, e o ar seco do sedimento. Adicionar 50 ul de solução de hibridação de 50, e incuba-se a 37 ° C durante 30 minutos para facilitar a dissolução. NOTA: sonda marcada podem ser armazenadas durante vários anos a -20 ° C. ATENÇÃO: 50 + solução de hibridização contém formamida, que é tóxico, pode irritar a pele, os olhos eo sistema respiratório, e também é um teratogênico. </li></ol> 4. Permeabilização, pré-tratamento e de hibridação de células fixas para Combined DNA-RNA FISH <ol><li> Rehidratar células fixadas em lamelas, por remoção de etanol a 70% e a adição da cultura de células de PBS para as placas de 6 poços. Incubar durante 5 min. Repita no total de três vezes. </li><li> Retirar a cultura de células de PBS, e adicionar 0,2% de pepsina para as placas de 6 poços, para permeabilizar as células. Incubar as placas de 6 poços em um banho de água a 37 ° C durante 4 min. Remover pepsina, e inativar com RNAse livre H 2 O. </li><li> Células pós-fixas por incubação com PFA a 4% / PBS durante 5 min à temperatura ambiente. Após FIXAÇÃOem, lavar duas vezes com PBS de cultura de células durante 5 min. </li><li> Desidratar as células por incubação com etanol a 70% durante 3 min, 90% de etanol durante 3 minutos e etanol a 100% durante 3 min. Transferência de lamelas de placas de 6 poços e lamelas secos ar por vários minutos. </li><li> Idade células por incubação de lamelas a 65 ° C durante 1 hora numa placa de calor. </li><li> Pré-sonda hibridizar FISH com rato Cot-1 DNA pela adição de cada lamela para ser analisado, 25 ul de solução de hibridação de 50, 0,5 mL rato Cot-1 DNA, 1 ul de sonda marcada com biotina Xist e 1 ul de digoxigenina sonda BAC rotulado detectar o cromossomo X. Misturar em vortex, e desnaturadas a 99 ° C durante 5 min. Incubar imediatamente a 37 ° C durante 45 minutos, para permitir que Cot-1 DNA que hibridizam para repetir sequências presentes nas sondas. </li><li> Após o envelhecimento, manchar 50 ul de tampão de desnaturação (consistindo de 70% tampão fosfato formamide/2x SSC/10 mM) numa lâmina de vidro, e colocar na parte superior com lamelas de células virada towards o tampão de desnaturação. Incubar a 65 ° C durante 2 min. </li><li> Adicionar lamelas de volta para placas de 6 poços e as células pós-fix, incubando em gelo frio etanol 70% por 5 min. </li><li> Desidratar as células por incubação subsequente em etanol a 70% durante 3 min, 90% de etanol durante 3 minutos e etanol a 100% durante 3 min. Remover lamelas de placas de 6 poços, e deixe secar por alguns minutos. </li><li> Manchar sonda pré-hibridizados em uma lâmina de vidro, e incubar lamela por cima. Colocar as lâminas em uma câmara humidificada cheio com 50 ml de 50% formamide/2x SSC, e incubar durante a noite a 37 ° C. </li></ol> 5. Lavagens pós-hibridação e o anticorpo de detecção da sonda mediada <ol><li> Preencha placas de 6 poços com 2x SSC, e pré-quente até 42 ° C. Adicionar lamelas após incubação durante a noite, e incuba-se a 42 ° C durante 5 min. </li><li> Retirar 2x SSC, e incubar lamelas com 50% formamide/2x SSC durante 10 min a 42 ° C. Repetir um total de 3 vezes (30 min de lavagem completamente). </li&gt; <li> Retirar 50% formamide/2x SSC, e lavagem das lâminas com Tris-salino-Tween (TST) em 2 x 5 min à temperatura ambiente. </li><li> Mancha 50 ul de Tris-salino-BSA (TSBSA) por lamela em novas lâminas de vidro, e incubar lamelas de cabeça para baixo para o bloqueio durante 30 minutos à temperatura ambiente numa câmara escura TST-humidificado. </li><li> Transferência de lamelas de volta para placas de 6 poços e lavar 2 x 5 min com TST. </li><li> Mancha 50 ul de anticorpo de ovelha anti-DIG (1:500 em TSBSA) por lamela em novas lâminas de vidro, e incubar lamelas de cabeça para baixo para o primeiro passo de incubação durante 30 minutos à temperatura ambiente numa câmara escura TST-humidificado. </li><li> Transferência de lamelas de volta para placas de 6 poços e lavar 2 x 5 min com TST. </li><li> Mancha 50 ul de anticorpo de coelho anti-ovelha conjugado com FITC (1:250 em TSBSA) por lamela em novas lâminas de vidro, e incubar lamelas de cabeça para baixo para o segundo passo de incubação durante 30 minutos à temperatura ambiente numa câmara escura TST-humidificada . </li><li> Transfer lamelas de volta para placas de 6 poços e lavar 2 x 5 min com TST. </li><li> Mancha 50 ul de anticorpo de cabra anti-coelho conjugado com FITC (1:250 em TSBSA) por lamela em novas lâminas de vidro, e incubar lamelas de cabeça para baixo para o terceiro passo de incubação durante 30 minutos à temperatura ambiente numa câmara escura TST-humidificada . </li><li> Transferência de lamelas de volta para placas de 6 poços e lavar 2 x 5 min com TST. </li><li> Mancha 50 ul de rato-anti-biotina anticorpo (1:200 em TSBSA) por lamela em novas lâminas de vidro, e incubar lamelas de cabeça para baixo para o quarto passo de incubação durante 30 minutos à temperatura ambiente numa câmara escura TST-humidificado. </li><li> Transferência de lamelas de volta para placas de 6 poços e lavar 2 x 5 min com TST. </li><li> Mancha 50 ul de anticorpo de burro anti-ratinho conjugado com rodamina (1:250 em TSBSA) por lamela em novas lâminas de vidro, e incubar lamelas de cabeça para baixo para o quinto passo de incubação durante 30 min à temperatura ambiente em um chamb escuro TST-humidificadoer. </li><li> Transferência de lamelas de volta para placas de 6 poços e lavar 2 x 5 min com TST. </li><li> Ponto 50 ul de anticorpo de cabra anti-cavalo conjugado com rodamina (1:250 em TSBSA) por lamela em novas lâminas de vidro, e incubar lamelas de cabeça para baixo para o sexto passo de incubação durante 30 minutos à temperatura ambiente em câmara escura TST-umidificado . NOTA: o anticorpo de cabra anti-cavalo vai reconhecer o anticorpo anti-burro-rato; quando disponível para o pesquisador, um anticorpo de cabra anti-burro vai funcionar tão bem. </li><li> Transferência de lamelas de volta para placas de 6 poços e lavar 2 x 5 min com TST. Lavar mais 5 min com Tris-salino (TS). </li><li> Desidratar as células por incubação subsequente em etanol a 70% durante 3 min, 90% de etanol durante 3 minutos e etanol a 100% durante 3 min. Remover lamelas de placas de 6 poços, e deixe secar por alguns minutos. </li><li> Manchar uma gota de meio de montagem com DAPI (ver reagentes) em uma lâmina de vidro, e adicionar lamela de cabeça para baixo em cima. Selar os slides com esmalte de unha eavaliar os resultados de FISH por microscopia fluorescente (Figura 1). </li></ol>

<ol>
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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>

Os autores não têm nada a revelar.

Combinado DNA-RNA FISH pode ser tecnicamente desafiadora, como condições adequadas para DNA FISH pode ser muito dura para moléculas de RNA menos estáveis. Várias abordagens têm sido aplicadas para estudar ambos o ADN e ARN na mesma célula, evitando a necessidade de incubação simultânea com as sondas de detecção, tanto o ADN e ARN. Por exemplo, numa abordagem de sobreposição, primeiro RNA FISH é realizada, e as células são gravadas, e as coordenadas sejam tomadas. Subsequentemente, os mesmos são utiliza...

Estudando as células e tecidos por meio de hibridação in situ fluorescente (FISH) tem, desde a sua introdução no final de 1970 um, permitiu aos pesquisadores estudar genes, organização da cromatina e expressão gênica em nível subcelular. PEIXES DNA é freqüentemente usada em citogenética, cariótipo 2, diagnósticos de câncer 3 e rastreamento genético pré-implantação 4, e tem um papel importante na investigação molecular 5-6, uma vez que permite a detecção de ácidos nucleicos em seu ambiente nativo. Análise de expressão de célula única de RNA FISH pode detectar transcrições primárias nativas e RNAs não-codificantes sendo transcrita de cromossomos, e oferece vantagens a outras técnicas que avaliam a expressão de genes em nível populacional, incluindo, por exemplo, RT-PCR quantitativo, o genoma análise de expressão larga ou Northern blotting . Ao visualizar transcrições de RNA originários diretamente de seus locais de origem, tem sido, por exemplo,notado que a expressão do gene é estocástico 7, e às vezes pode ser alelo específico 8. Melhorias na técnica têm ainda permitiu a detecção e quantificação de moléculas de mRNA de células individuais dentro de 9-11. O princípio básico de FISH consiste de hibridização de ácidos nucleicos no interior da célula a uma sonda de ácido nucleico, por meio de altamente específica de Watson e Crick emparelhamento de bases. A sonda pode estar directa ou indirectamente detectado, resultando num sinal que pode ser visualizado microscopicamente. As tentativas iniciais consistiam de sondas radioativamente marcados, que tiveram inconvenientes com base em questões de segurança, limitada resolução espacial ea capacidade de detectar um único alvo de cada vez 12-14. O subsequente desenvolvimento de marcadores não radioactivos, incluindo fluorocromos, haptenos e enzimas, permitiu o uso generalizado de FISH como uma técnica de biologia molecular de rotina. A sonda de FISH pode ser diretamente rotulados com fluorochromes por ligação química de moléculas fluorescentes para sequências de ácido nucleico e 15 a integração de nucleótidos marcados com fluorescência 16-19, ou a sonda pode ser indirectamente visualizadas após integração de haptenos (incluindo biotina e digoxigenina) e detecção imunológica de haptenos por anticorpos específicos de hapteno conjugado com repórter fluorescente moléculas 20-21. A última abordagem permite a amplificação do sinal, utilizando várias camadas de anticorpos marcados com fluorescência que são usados ​​para melhorar o sinal original, e permite a detecção de espécies de ARN que são expressos em níveis baixos. Através da combinação de técnicas de rotulagem diretos e indiretos e vários haptenos, vários alvos podem ser visualizadas simultaneamente na mesma célula. Um dos passos mais importantes protocolos de peixe é a hibridização da sonda ao seu alvo. Embora, em teoria, uma sonda vai ligar especificamente apenas com o seu alvo, na prática, esta especificidadenão é sempre conseguido, como sondas podem ligar-se a regiões homólogas, e as condições de hibridização não será sempre ideal para uma determinada espécie de região de DNA ou de RNA. As lavagens pós-hibridação são, portanto, de particular importância, uma vez que podem aumentar o rigor do procedimento FISH, e pode impedir a ligação não específica de sondas FISH, o que resultaria num elevado nível de ruído de fundo. Como moléculas de ARN são de cadeia simples que pode ser facilmente hibridado com uma sonda de FISH. Em contraste, a molécula de ADN de cadeia dupla deve em primeiro lugar um passo de desnaturação, após o que uma sonda pode hibridizar. Isto é geralmente conseguido por meio de aquecimento da amostra, o que resulta em desnaturação do ADN. No entanto, sob essas condições adversas, frágeis, único moléculas de ARN de cadeia pode ser perdida. Portanto, o ADN-ARN combinados FISH requer optimização significativo das condições, e é tecnicamente mais difícil em comparação com a detecção separada de apenas RNA ou DNA. Aqui nós apresenta protocolo detalhado do combinado, simultânea FISH DNA-RNA, que nos permitiu estudar inativação do cromossomo X (ICX) na diferenciação de rato fêmea células-tronco embrionárias 22-24. XCI é um mecanismo epigenético crucial para o desenvolvimento embrionário fêmea 25, e os resultados em heterochromatinization e, portanto, o silenciamento de um dos dois cromossomas X em indivíduos do sexo feminino 26-27. Essencial para esse processo é a não-codificante RNA Xist 28-30, que é regulada pelos 24 proteínas RNF12 22-23 e Rex1. Expressão Xist torna-se regulada no futuro cromossomo X inativo (Xi) durante o desenvolvimento embrionário ou a diferenciação de células ES in vitro , e pode se espalhar ao longo do cromossomo X e, assim, atrair cromatina remodelação enzimas que resultam na paralisação da transcrição do cromossomo X 31. Esta propagação de Xist ARN pode ser visualizado por FISH ARN como revestimento de cromo Xalguns, que também é referida como uma nuvem Xist. Como as células ES femininos pode perder um de seus cromossomos X, devido à instabilidade genômica, nós e outros têm utilizado combinado DNA-RNA FISH para estudar XCI, para certificar-se de que as células só cariótipo estáveis ​​são avaliados na análise deste importante processo 22,32 -34. Tal como acontece com todas as técnicas de biologia molecular, vários protocolos diferentes excelentes foram publicados 35-38. Aqui apresenta-se a método, a partir da indução de diferenciação em células ES de ratinho, a fixação das células, a rotulagem de sondas FISH por Nick-translation, pré-tratamento de células fixas para permitir a permeabilização e subsequente absorção de sonda, a hibridação da sonda com o alvo, e, finalmente, a detecção da sonda por meio de anticorpos marcados com fluorescência. O protocolo aqui apresentado permite a detecção fiel de Xist RNA eo cromossomo X, no prazo de dois dias, e os princípios básicos desta técnica pode provavelmente ser adaptado para otseus sistemas e áreas de pesquisa.

<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

Hibridização in situ fluorescente (FISH) é uma técnica molecular que permite a detecção de ácidos nucleicos em células. PEIXES DNA é frequentemente usado em citogenética e diagnóstico de câncer, e pode detectar aberrações do genoma, que muitas vezes tem importantes implicações clínicas. ARN FISH pode ser utilizado para detectar as moléculas de RNA nas células e proporcionou perspectivas importantes na regulação da expressão do gene. Combinando DNA e RNA FISH dentro da mesma célula é tecnicamente desafiador, como condições adequadas para DNA FISH pode ser muito dura para frágeis, único moléculas de ARN de cadeia. Temos aqui apresentam um protocolo facilmente aplicável, que permite que o conjunto, a detecção simultânea de Xist RNA e DNA codificado pelos cromossomas X. Este protocolo FISH DNA-RNA combinado pode provavelmente ser aplicada a outros sistemas em que tanto o ARN e ADN precisam ser detectado.

Usando o protocolo acima mencionado para combinado ADN-ARN FISH, temos sido capazes de visualizar X inactivação do cromossoma na diferenciação de células estaminais embrionárias fêmeas. Figura 1 mostra um exemplo representativo de uma experiência FISH de ADN-ARN, em que foram detectados tanto Xist (que é visível como uma chamada Xist RNA nuvem no cromossomo X inativo e um pontinho de transcrição basal no cromossomo X ativo), e uma região do cromossomo X, que é visível co...

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

Hibridização in situ fluorescente (FISH), permite a detecção de ácidos nucleicos no seu ambiente nativo dentro das células. Nós aqui descrever um protocolo para o combinado, a detecção simultânea de RNA e DNA, por meio de FISH, que pode ser usado para estudar inativação do cromossomo X em células-tronco embrionárias.

DNA-RNA combinado fluorescente<em&gt; Em situ</em&gt; A hibridização (FISH) para estudar inativação do cromossomo X em Diferenciada Feminino Rato Células-Tronco Embrionárias

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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27.5K visualizações.  Erasmus MC - University Medical Center.  Hibridização in situ fluorescente (FISH), permite a detecção de ácidos nucleicos no seu ambiente nativo dentro das células. Nós aqui descrever um protocolo para o combinado, a detecção simultânea de RNA e DNA, por meio de FISH, que pode ser usado para estudar inativação do cromossomo X em células-tronco embrionárias. 

Vídeo: 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 Whole Mount Alta Resolução Duplo Fluorescente In Situ Hibridização

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

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.

Fluorescência In situ Hybridization (FISH) Protocolo no esperma humano

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.

Isolamento e derivação de células embrionárias de rato 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.

Monte todo In Situ Hibridação de E8.5 de Embriões E11.5 rato

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.

Detecção de RNA viral por fluorescência<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.

Análise de uma única célula de transcrição do gene pela RNA Fluorescente<em&gt; In Situ</em&gt; A hibridização (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.

Tempo cromossomo Replicando Combinado com Fluorescente<em&gt; Em situ</em&gt; Hibridização

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.

Todo o Monte RNA Fluorescente<em&gt; In situ</em&gt; A hibridação de<em&gt; Drosophila</em&gt; Embriões

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).

Imunofluorescência combinados e FISH DNA em 3D preservado núcleos interfásicos para estudar mudanças na organização nuclear 3D

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.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<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 ...