Queremos agradecer aos membros do Brown e laboratórios Lam pela sua participação na crítica a este vídeo e artigo. Este trabalho foi financiado por fundos do Canadian Institutes for Health Research eo Michael Smith Fundação para a Pesquisa em Saúde.

EXTRAÇÃO DE DNA E PREPARAÇÃO DA AMOSTRA DNA de uma variedade de diferentes amostras (células cultivadas fresca, congelada, bem como tecidos fixados em formalina incluído em parafina) podem ser usados ​​para MEDIP. É importante o uso de DNA altamente purificado, sem proteínas associadas, tais como histonas. Também é importante para remover RNA, tanto quanto possível a partir da amostra, como ele pode interferir tanto com a quantificação de DNA e ligação aos anticorpos. A quantidade de DNA usado para MEDIP pode variar de 200 ng de 1 mg, dependendo da quantidade de DNA disponíveis. Para demonstrar este protocolo, 1 mg de DNA será usado. O seguinte protocolo irá proporcionar alta qualidade do DNA fita dupla a partir de células cultivadas. Outros protocolos devem ser empregados para a extração de DNA de tipos de amostra outro. <ol><li> Adicionar 400 mL de tampão de digestão para um pellet de células em um tubo Eppendorf 1,7 ml. </li><li> Adicionar 100 mg de proteinase K para o tubo e incubar durante a 50 ° C. </li><li> Adicionar 500 mL de fenol pH 7 e misturar suave mas completamente por inversão. </li><li> Giram a 13 000 g durante 10 minutos em temperatura ambiente. </li><li> Remover fração (em cima) aquosa para um novo tubo. </li><li> Repita as etapas 3 a 5 uma vez. </li><li> Adicionar 500 mL pH 01:01 fenol / clorofórmio 7 e misturar suave mas completamente por inversão. </li><li> Giram a 13 000 g durante 10 minutos em temperatura ambiente. </li><li> Remover fração (em cima) aquosa para um novo tubo. </li><li> Adicionar 40 mg de RNase A e incubar 1 hora a 37 ° C. </li><li> Adicionar 500 mL de fenol pH 7 e misturar suave mas completamente por inversão. </li><li> Giram a 13 000 g durante 10 minutos em temperatura ambiente. </li><li> Remover fração (em cima) aquosa para um novo tubo. </li><li> Repita as etapas 11 a 13 uma vez. </li><li> Adicionar 500 mL pH 01:01 fenol / clorofórmio 7 e misturar suave mas completamente por inversão. </li><li> Giram a 13 000 g durante 10 minutos em temperatura ambiente. </li><li> Remover fração (em cima) aquosa para um novo tubo. </li><li> Adicionar volume 1/10th 3 M acetato de sódio (40 mL) e misture bem. </li><li> Adicionar 2 volumes (900 L) de etanol 100%, misture bem e coloque a -20 ° C por 20 minutos. </li><li> Giram a 13 000 g durante 20 minutos a 4 ° C. </li><li> Remover o etanol, spin pulso, e remover o etanol residual. </li><li> Adicionar 500 mL de etanol frio a 70% de lavar. Giram a 13 000 g durante 20 minutos a 4 ° C. </li><li> Remover o etanol 70%, giro de pulso, e remover o etanol residual por pipetagem. </li><li> O ar seco do sedimento com a tampa aberta por 10 minutos em temperatura ambiente para remover todos os vestígios do etanol residual. </li><li> Ressuspender o DNA em 50 ul de dH2O esterilizado durante a noite a 4 ° C. </li><li> Quantificar o DNA utilizando um Espectrofotômetro NanoDrop. Um A 260: Uma relação de 280 de 1.8 é ideal. </li><li> Determinar a qualidade do DNA e faixa de tamanho em um gel de agarose com 100 bp ladder. Tão pouco como 10 ng de DNA genômico pode ser executado em um gel de agarose 1,7% seguido de coloração com um corante que é altamente sensível a pequenas quantidades de DNA, tais como ouro SYBR. </li><li> Em um tubo siliconizado por amostra, prepare 1 mg de DNA em um volume total de 50 l com o restante do volume composto esterilizado com dH 2 O. </li></ol> DNA sonicação DNA sonicação eo protocolo MEDIP deve ser realizada em tubos siliconzied para evitar a ligação específica de proteínas não às paredes do tubo. Vezes sonicação ideal para as amostras de DNA são baseados no grau de fragmentação da amostra de DNA determinado a partir de eletroforese em gel (Passo 27). Por exemplo, o DNA extraído de células em cultura deve ser de muito alto peso molecular, e, posteriormente, exigir mais do que sonicação DNA extraído de amostras de arquivo que são muitas vezes parcialmente degradada. Se as amostras são de uniformemente elevado peso molecular, é razoável neste momento a proceder à sonicação, conforme descrito neste protocolo sem verificar cada amostra individualmente. Se as amostras são fragmentados com amostras de arquivo, será necessário adaptar o procedimento de sonicação provavelmente por diminuir os tempos de sonicação para obter 300-100bp fragmentos. Se você esperar para processar as amostras que são parcialmente degradadas, otimização de parâmetros de sonicação pode ser realizada em uma amostra representativa daqueles de interesse. Com base no grau de fragmentação do DNA observados de eletroforese em gel (Passo 27), o experimentador pode predeterminar o tempo de sonicação ideal para a amostra. Aqui nós descrevemos um método para obter 300-1000 fragmentos de DNA bp por sonicação com um dispositivo automatizado sonicando (Bioruptor de Diagenode, UCD-200 TM) usando DNA de alto peso molecular. <ol><li> Água em Biorupter deve ser a 4 ° C. </li><li> Sonicate por 7 minutos em configurações automáticas (30 seg em 30 seg off à potência máxima). </li><li> Remover 800 ng (40 L) de produto sonicado e colocar em tubo de centrífuga siliconizado 1,7 ml para a imunoprecipitação (IP) da reação. </li><li> Retiradas restantes ng 200 (10 ml) para servir como entrada (IN) de ADN de referência (loja a 4 ° C). </li&gt; </ol> IMMUNOPRECIPITATON de DNA metilado <ol><li> Desnaturar o DNA que será usado para a reação IP (800 ng) a 95 ° C por 10 minutos em banho-maria. </li><li> Arrefecer imediatamente no gelo. Deixe esfriar completamente DNA (aproximadamente 5 minutos no gelo) antes de prosseguir com o próximo passo. </li><li> Adicionar 5 mg de anticorpos monoclonais. </li><li> Adicionar tampão IP (usado em temperatura ambiente durante todo este protocolo) para um volume final de 500 mL. </li><li> Incubar por 2 horas a 4 ° C na rotação titular tubo. </li><li> Pouco antes de o passo 5 está completa, prepare Dynabeads por lavagem (Passos 6-11). Em primeiro lugar, voltar a suspender as contas completamente dentro do frasco em vortex. </li><li> Transferência de 30 mL (~ 2 x 10 7) de Dynabeads ressuspenso por reação mais 1, em um novo tubo siliconizado (por exemplo, se fazendo 8 reações, remova contas suficiente para 9, ou seja, 270 mL). </li><li> Colocar o tubo na cremalheira magnética por 2 minutos em temperatura ambiente. </li><li> Pipeta fora do sobrenadante. Quando retirar o sobrenadante, evite tocar os grânulos contra a parede interna (onde as contas atrair para o ímã) com a ponta da pipeta. </li><li> Remova o tubo do ímã, e ressuspender as contas em um excesso de volume de tampão IP (750 mL mL-1000). Colocar o tubo de volta na prateleira magnética por 2 minutos em temperatura ambiente. </li><li> Repita o lava mais uma vez, e depois voltar a suspender as contas lavados em tampão IP no volume original removidos na Etapa 7. </li><li> Adicionar 30 mL de Dynabeads lavada para cada reação IP </li><li> Incubar em um suporte de tubo rotativo durante 2 horas a 4 ° C. </li><li> Após a incubação estiver concluída, coloque o tubo na cremalheira magnética por 2 minutos em temperatura ambiente. </li><li> Pipeta fora do sobrenadante. Evite tocar a parede interior do tubo (onde as contas atrair para o ímã) com a ponta da pipeta. Adicionar 500 mL de tampão IP. Misture o conteúdo do tubo e colocá-lo de volta na prateleira magnética por 2 minutos. Repita lave com 500 ul de buffer IP uma só vez. </li><li> Após a remoção do sobrenadante da última lavagem, ressuspender as contas em 400 mL de tampão de digestão. </li><li> Tratar a reação com 100 mg de Proteinase K e incubar durante a 50 ° C. </li></ol> PURIFICAÇÃO DE DNA IMMUNOPRECIPITATED <ol><li> Adicionar 500 mL pH 01:01 fenol / clorofórmio 7 e vortex completamente. </li><li> Giram a 13 000 g durante 10 minutos em temperatura ambiente. </li><li> Remover fração (em cima) aquosa para um novo tubo. </li><li> Repita as etapas 1 a 3 se a interfase entre as camadas aquosa e orgânica parece nublado. </li><li> Adicione 1 / 10 º volume 3 M acetato de sódio (40 mL) e vortex. </li><li> Adicionar 1 ml de glicogênio (20 mcg / mL) e vortex. </li><li> Adicionar 2 volumes (1000 L) de etanol 100%, vortex, e coloque a -20 ° C por 20 minutos. </li><li> Giram a 13 000 g durante 20 minutos a 4 ° C. </li><li> Remover o etanol, spin pulso, e remover o etanol residual. </li><li> Adicionar 500 mL de etanol frio a 70% de lavar. Brevemente Vortex, e giram a 13 000 g durante 20 minutos a 4 ° C. </li><li> Remover o etanol 70%, giro de pulso, e remover o etanol residual por pipetagem. </li><li> O ar seco do sedimento com a tampa aberta por 10 minutos em temperatura ambiente para remover todos os vestígios do etanol residual. </li><li> Ressuspender DNA sedimento em 10 ml esterilizados dH 2 0. </li></ol> Validação por PCR <ol><li> Você pode testar para garantir que o seu procedimento MEDIP está funcionando, a execução do protocolo MEDIP usando DNA humano normal (masculino ou feminino) e, posteriormente, assaying uma região conhecida por ser enriquecido para metilação. </li><li> Remover 30% do produto MEDIP ao tubo de PCR, e outros 30% do produto MEDIP para outro tubo de PCR. </li><li> Colocar 10 ng de DNA IN em um tubo de PCR, e 10 ng de DNA IN em outro tubo de PCR. Você deve ter agora quatro reações distintas PCR para configurar. </li><li> Realizar PCR usando primers H19 e CTRL como indicado na Tabela 1. </li><li> Prepare duas mastermixes (um para cada conjunto de primers) para reações de PCR com 12,5 mL de volume total, como descrito na Tabela 2. </li><li> Thermocycle do PCR usando as condições descritas na Tabela 3. </li><li> Correr 5 mL de produtos de PCR em um gel de agarose 2% para visualização. </li><li> Os resultados esperados para MEDIP sucesso são apresentados na Tabela 4. </li></ol> Tabelas Tabela 1: H19 e CTRL primers para a validação de PCR. <table border="1"><tbody><tr><td> Definir Primer </td><td> Primer para a frente </td><td> Primer reverso </td><td> Tamanho do produto antecipado </td></tr><tr><td> H19 </td><td> H19_F 5&#39;-cgagtgtgcgtgagtgtgag </td><td> H19_R 5&#39;-ggcgtaatggaatgcttgaa </td><td> 174 bp </td></tr><tr><td> CTRL (controle) </td><td> CTRL_F5&#39;-gagagcattagggcagacaaa </td><td> CTRL_R 5&#39;-gttcctcagacagccacattt </td><td> 139 bp </td></tr></tbody></table> Tabela 2. Mastermixes para as reações PCR. <table border="1"><tbody><tr><td></td><td> H19 Mix (por Rxn) </td><td> CTRL Mix (por Rxn) </td></tr><tr><td&gt; 2 O DDH </td><td> 6,875 </td><td> 6,875 </td></tr><tr><td> Tampão 10X </td><td> 1,25 </td><td> 1,25 </td></tr><tr><td> dNTP mix (10 mM cada) </td><td> 0,25 </td><td> 0,25 </td></tr><tr><td> MgCl 2 (50 mM) </td><td> 0,25 </td><td> 0,25 </td></tr><tr><td> Primers (H19_F / R ou CTRL_F / R) (10 mM cada F / R) </td><td> 0,625 </td><td> 0,625 </td></tr><tr><td> Taq Platinum </td><td> 0,25 </td><td> 0,25 </td></tr></tbody></table> Tabela 3. Condições de PCR thermocyling. <table border="1"><tbody><tr><td></td><td> 95 ° C </td><td> 05:00 </td></tr><tr><td rowspan="3"> 40X </td><td> 95 ° C </td><td> 00:30 </td></tr><tr><td> 56 ° C </td><td> 00:30 </td></tr><tr><td> 72 ° C </td><td> 00:15 </td></tr></tbody></table> Tabela 4. Resultados esperados para o MEDIP PCR bem sucedido. <table border="1"><tbody><tr><td></td><td colspan="2" align="center"> DNA modelo </td></tr><tr><td> Primer usados </td><td> IN </td><td> IP </td></tr><tr><td> H19 </td><td> Positivo </td><td> Positivo </td></tr><tr><td> CTRL </td><td> Positivo </td><td> Negativo </td></tr></tbody></table>

<ol>
	<li>Beck, S., Rakyan, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+methylome:+approaches+for+global+DNA+methylation+profiling.">The methylome: approaches for global DNA methylation profiling.</a> Trends Genet. 24, 231-237 (2008).</li><li>Lu, Q., 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=Epigenetics,+disease,+and+therapeutic+interventions.">Epigenetics, disease, and therapeutic interventions.</a> Ageing research reviews. 5, 449-467 (2006).</li><li>Zilberman, D., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+DNA+methylation+patterns.">Genome-wide analysis of DNA methylation patterns.</a> Development. 134, 3959-3965 (2007).</li><li>Feinberg, A. P., Tycko, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+of+cancer+epigenetics.">The history of cancer epigenetics.</a> Nature reviews. 4, 143-153 (2004).</li><li>Weber, 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=Distribution,+silencing+potential+and+evolutionary+impact+of+promoter+DNA+methylation+in+the+human+genome.">Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome.</a> Nat Genet. 39, 457-466 (2007).</li><li>Weber, 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=Chromosome-wide+and+promoter-specific+analyses+identify+sites+of+differential+DNA+methylation+in+normal+and+transformed+human+cells.">Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells.</a> Nat Genet. 37, 853-862 (2005).</li><li>Wilson, I. 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=Epigenomics:+mapping+the+methylome.">Epigenomics: mapping the methylome.</a> Cell Cycle. 5, 155-158 (2006).</li><li>Gazin, C., Wajapeyee, N., Gobeil, S., Virbasius, C. M., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+elaborate+pathway+required+for+Ras-mediated+epigenetic+silencing.">An elaborate pathway required for Ras-mediated epigenetic silencing.</a> Nature. 449, 1073-1077 (2007).</li><li>Karpinski, P., Sasiadek, M. M., Blin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+epigenetic+patterns+in+the+etiology+of+gastrointestinal+cancers.">Aberrant epigenetic patterns in the etiology of gastrointestinal cancers.</a> Journal of applied. 49, 1-10 (2008).</li><li>Maekawa, M., Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics:+relations+to+disease+and+laboratory+findings.">Epigenetics: relations to disease and laboratory findings.</a> Current medicinal chemistry. 14, 2642-2653 (2007).</li><li>Vucic, E. A., Brown, C. J., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+of+cancer+progression.">Epigenetics of cancer progression.</a> Pharmacogenomics. 9, 215-234 (2008).</li><li>Egger, G., Liang, G., Aparicio, A., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+in+human+disease+and+prospects+for+epigenetic+therapy.">Epigenetics in human disease and prospects for epigenetic therapy.</a> Nature. 429, 457-463 (2004).</li><li>Jones, P. A., Baylin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+role+of+epigenetic+events+in+cancer.">The fundamental role of epigenetic events in cancer.</a> Nat Rev Genet. 3, 415-428 (2002).</li><li>Fraga, M. F., Esteller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation:+a+profile+of+methods+and+applications.">DNA methylation: a profile of methods and applications.</a> Biotechniques. 33, 632-649 (2002).</li></ol>

Há uma consciência crescente do papel significativo desempenha na doença de metilação do DNA, portanto, o desenvolvimento de ensaios para medir esta modificação estão se tornando cada vez mais importante 3, 12, 13. A técnica MEDIP é um instrumento favorável para triagem, tanto do genoma inteiro-o e locus específico de nível 6, 7. Esta técnica oferece uma visão rápida dos níveis de metilação do DNA usando quantidades limitadas de partida DNA e permite comparações fáceis entre d...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Biorupter sonicator</td>
<td>Tool</td>
<td>Diagenode</td>
<td>UCD-200 TM</td>
<td>&nbsp;</td>
<tr>
<td>1.7ml SafeSeal Microcentrifuge Tubes</td>
<td>Other</td>
<td>Sorenson BioScience</td>
<td>11510</td>
<td>&nbsp;</td>
<tr>
<td>ND 3300 Spectrophotometer</td>
<td>Tool</td>
<td>NanoDrop</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Primary Antibody: Anti-5'-methylcytosine mouse mAb</td>
<td>Reagent</td>
<td>CalBiochem</td>
<td>162 33 D3</td>
<td>&nbsp;</td>
<tr>
<td>Secondary Antibody: Dynabeads M-280 Sheep anti-mouse IgG</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>112-01D</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Tube Rack</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>CS15000</td>
<td>&nbsp;</td>
<tr>
<td>Mini LabRoller</td>
<td>Tool</td>
<td>Labnet International</td>
<td>H5500</td>
<td>&nbsp;</td>
<tr>
<td>IP Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100. Stored at room temperature</td>
</tr>
<tr>
<td>Digestion Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM Tris pH8.0, 100 mM EDTA, 0.5% SDS, 50 mM NaCl</td>
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methylated dna immunoprecipitation

A identificação de padrões de metilação do DNA é um procedimento comum no estudo da epigenética, como a metilação é conhecido por ter efeitos significativos sobre a expressão gênica, e está envolvida com desenvolvimento normal, bem como doença<sup&gt; 04/01</sup&gt;. Assim, a capacidade de discriminar entre o DNA metilado e não metilado-DNA é essencial para a geração de perfis de metilação para esses estudos. Metilado DNA imunoprecipitação (MEDIP) é uma técnica eficiente para a extração de DNA metilado partir de uma amostra de interesse<sup&gt; 07/05</sup&gt;. Uma amostra de tão pouco como 200 ng de DNA é suficiente para o anticorpo, ou imunoprecipitação (IP), a reação. DNA é sonicado em fragmentos que variam em tamanho de 300-1000 pb, e está dividido em immunoprecipitated (IP) e de entrada (IN) parcelas. DNA IP é posteriormente calor desnaturado e, em seguida, incubadas com anti-5&#39;mC, permitindo que o anticorpo monoclonal de se ligar DNA metilado. Após isso, esferas magnéticas contendo um anticorpo secundário com afinidade para o anticorpo primário são adicionados e incubados. Estes anticorpos bead-linked ligará o anticorpo monoclonal utilizado na primeira etapa. DNA ligado ao complexo de anticorpos (DNA metilado) é separado do resto do DNA usando um ímã para puxar os complexos de solução. Várias lavagens com tampão IP são, então, realizada para remover o desacoplado, DNA não-metilado. O DNA metilado / anticorpo complexos são então digeridos com proteinase K para digerir os anticorpos, deixando apenas o DNA metilado intacta. O DNA enriquecido é purificado por fenol: clorofórmio extração para remover a matéria de proteínas e, em seguida, precipitado e ressuspenso em água para uso posterior. Técnicas de PCR pode ser usado para validar a eficiência do procedimento MEDIP pela análise dos produtos de amplificação de IP e no DNA das regiões conhecidas por falta e conhecido por conter seqüências metiladas. O DNA purificado metilado pode então ser usada para o locus-específica (PCR) ou genome-wide (microarray e seqüenciamento) Estudos de metilação, e é particularmente útil quando aplicado em conjunto com outras ferramentas de pesquisa, tais como perfil de expressão gênica e hibridização genômica comparativa de matriz ( CGH)<sup&gt; 8</sup&gt;. Uma investigação mais aprofundada para metilação do DNA levará à descoberta de novos alvos epigenéticos, que por sua vez, pode ser útil no desenvolvimento de novas ferramentas de pesquisa terapêutica ou prognóstico para doenças como o câncer que se caracterizam por DNA metilado aberrantemente<sup&gt; 2, 4, 9-11</sup&gt;.

Watch this Scientific Journal Video about Methylated DNA Immunoprecipitation at JoVE.com

Methylated DNA Immunoprecipitation

Este vídeo demonstra o protocolo para imunoprecipitação DNA metilado (MEDIP). MEDIP é um procedimento dois dias que, seletivamente, extratos de fragmentos de DNA metilado partir de uma amostra do DNA genômico utilizando anticorpos com especificidade para 5-metilcitosina (anti-5 mC).

Imunoprecipitação DNA metilado

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

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23.2K visualizações.  BC Cancer Research Centre. Este vídeo demonstra o protocolo para imunoprecipitação DNA metilado (MEDIP). MEDIP é um procedimento dois dias que, seletivamente, extratos de fragmentos de DNA metilado partir de uma amostra do DNA genômico utilizando anticorpos com especificidade para 5-metilcitosina (anti-5 mC).

Vídeo: Methylated DNA Immunoprecipitation

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Cromatina imunoprecipitação de células estaminais embrionárias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Biologia

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Perfil de anticorpos por sistemas de imunoprecipitação luciferase (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA-Stable Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

FCM-IP: Imunoprecipitação Detectado por citometria de fluxo

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Ensaio para imunoprecipitação da cromatina Genes Tissue-específicos usando embriões em início de carreira do rato

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Cromatina imunoprecipitação (CHIP), utilizando Drosophila Tecido

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Imunoprecipitação da Cromatina eficiente usando Limitando quantidades de biomassa

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Geração de alta Chromatin Template DNA Immunoprecipitation Qualidade para seqüenciamento de alto rendimento (CHIP-seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imagens em tempo real de uma única Engineered RNA transcritos em células vivas usando raciométrica Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

De gravidade reduzida Manifestações ambiente de hardware de uma tecnologia de mistura de fluxo Cytometer e Companion microfluídicos Prototype Miniaturized

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...