Agradecemos T. Hung, MC. Tsai, O. Manor, E. Segal, M. Kuroda, T. Swigut, e I. Shestopalov para as discussões. Apoiado pela Agência de Ciência, Tecnologia e Pesquisa de Cingapura (CC), NIH-R01 e R01 CA118750-HG004361 (HYC), e do Instituto de Medicina Regenerativa da Califórnia (HYC). HYC é um cientista de carreira precoce do Howard Hughes Medical Institute.

1. Projeto Sonda Design DNA anti-senso de lado a lado sondas para a recuperação seletiva de RNA alvo por piar. <ol><li> Projeto sondas anti-senso oligo usando o designer sonda on-line em <a href="http://singlemoleculefish.com">singlemoleculefish.com</a> 18. </li><li> Use esses parâmetros: número de sondas = 1 / sonda de 100 pb de comprimento RNA, 2) Alvo% GC = 45, comprimento Espaçamento 4) = 60-80; 3) Comprimento Oligonucleotide = 20. Quebre RNA em segmentos se muito tempo para o designer. Omitir regiões de repetições ou homologia extensa. </li><li> Ordem sondas anti-senso de DNA com BiotinTEG em 3-primeiro-final. </li><li> Sondas etiqueta de acordo com as suas posições ao longo do RNA. Separá-los em dois grupos para que a piscina &quot;, mesmo&quot; contém todas as sondas de numeração 2, 4, 6, etc, e na piscina &quot;estranho&quot; contém sondas de numeração 1, 3, 5, etc Diluir pool de sondas para concentração de 100 mM e armazenar a -20 ° C. </li><li> Todas as experiências são para ser feita usandoambas as caixas, que servem como controles internos para o outro. Sinal dependente de ARN real estaria presente em ambos os pools, enquanto sonda específicas ruídos seria único para cada pool. Isto aplica-se tanto para CHIRP-qPCR e CHIRP-seq. </li></ol> 2. Células Colheita Recolher células que serão usados ​​para a experiência CHIRP. <ol><li> Crescer as células em placas de cultura de tecidos ou frascos até à confluência. Lavar com salina tamponada com fosfato (PBS) e uma vez trypsinize. Têmpera de tripsina com&gt; volume 2x de mídia, pipeta cima e para baixo para retirar as células e ressuspender em suspensão de células individuais. Transferir todos os meios e as células ressuspensas em tubos de 50 ml Falcon. 20 milhões de células são tipicamente suficiente para uma amostra CHIRP. </li><li> Girar em células 800RCF durante 4 min. Meios aspirado e ressuspender 40 milhões de células em 40 ml de PBS, combinar os tubos, se necessário. Girar em células 800RCF durante 4 min. Decantar PBS, aspirar cuidadosamente sobre um ângulo que o líquido restante. </li></ol&gt; 3. Cross-link células e recolher pellet celular Crosslink coletadas células com glutaraldeído para preservar RNA-cromatina interações e preparar pellet celular. <ol><li> Execute todas as etapas à temperatura ambiente. </li><li> Prepare glutaraldeído 1% em PBS à temperatura ambiente. Preparar 10 ml por 10 milhões de células (0,4 ml de caldo de glutaraldeído a 25% + 9,6 mL de PBS). O glutaraldeído deve ser utilizado fresco. </li><li> Toque fundo de tubos Falcon de desalojar pelotas. Ressuspender o sedimento de células em glutaraldeído a 1%, começando com um pequeno volume para evitar pedaços, em seguida, o início até ao volume total. Inverta para misturar. Reticulação durante 10 min à temperatura ambiente num agitador de ponta a ponta ou rotador. </li><li> Extingue-se a reacção de reticulação com um volume de 1/10th de 1,25 M de glicina, à temperatura ambiente durante 5 min. </li><li> Girar em 2000RCF durante 5 min. Aspirar o sobrenadante e lava-pelete com 20 mL de PBS gelado uma vez, girando a 2000RCF durante 5 min. </li><li> Aspirar e ressuspender o wincinerada, reticulada pelete com 1 ml de PBS, refrigerada por 20 milhões de células. Transfira cada ml a um tubo Eppendorf e giram a 2000RCF durante 3 min a 4 ° C. Remover PBS, tanto quanto possível, com a ponta da pipeta com cuidado. </li><li> Flash-congelar os peletes de células em azoto líquido e armazenada a -80 ° C indefinidamente. </li></ol> 4. Lise Celular Lisar células reticulados para preparar lisado celular. <ol><li> Descongelar sedimentos celulares congelados à temperatura ambiente. Toque difícil desalojar e misturar o pellet celular. Girar para baixo o sedimento em 2000RCF durante 3 min a 4 ° C. Use uma forte ponta da pipeta 10 ul para remover qualquer PBS restante. </li><li> Em uma balança eletrônica (precisão de 1 mg) tara de um tubo de Eppendorf vazio (os nossos tubos de pesar 1.060 gramas muito consistente). Pese cada pellet e gravar o seu peso. Um prato centímetros completa 15 de células HeLa reticulados tipicamente pesa 100 mg. </li><li> Tampão de Lise suplemento (massa 10X de pellet, por exemplo, 1 ml para 100 mg) com fresh inibidor da protease, PMSF e Superase-in (veja a lista de tampão em anexo). Misture bem. </li><li> Adicionar 10X volume de Tampão de Lise completado, para cada tubo e ressuspender o sedimento. Para as pelotas pequenas &lt;25 mg, ressuspender em 250 ul de tampão de lise suplementado. Suspensão deve ser suave. Se não, dividir suspensão em 500 uL alíquotas e usar uma motorizada pelete misturador para quebrar grumos. Proceder de imediato à sonicação. </li></ol> 5. Sonicação DNA de cisalhamento por sonicando lisados ​​de células reticuladas. <ol><li> Sonicar lisado celular em Bioruptor em 15 tubos Falcon ml. Use &lt;1,5 lisado ml em cada tubo, e para mais rapidamente sonicação, sonicar não mais do que dois tubos de cada vez. </li><li> Sonifique em um banho de água 4 ° C a mais alta definição com 30 segundos, 45 segundos OFF intervalos de pulso. Verifique lisado cada 30 min. Continuar sonicando até que o ligado celular não é mais turvo. Isso pode demorar tão pouco como 30 minutos e até 4 horas. O númerode tubos, o volume da amostra, a temperatura do banho, eo período de tempo de sonicação irá afectar o tempo que o processo leva. Tubos provavelmente sonicar em taxas diferentes, de modo juntam-los em conjunto a cada 30 min e redistribuir em tubos originais para assegurar a homogeneidade. Nota: o glutaraldeído-reticulado células demorar muito mais para sonicate do que seus equivalentes de formaldeído. </li><li> Quando lisado transforma claro, transferir 5 uL lisado para um novo tubo Eppendorf. Adicione 90 uL de DNA de proteases K (PK) Buffer (ver lista buffer) e 5 PK uL. Vortex para misturar e girar rapidamente. Incubar durante 45 min a 50 ° C. </li><li> Extrair DNA com Qiagen kit de purificação PCR. Eluir o DNA em 30 uL Tampão eluição Qiagen (EB) e verificar o tamanho do DNA em 1% gel de agarose. Se grandes quantidades de DNA é o esfregaço 100-500 pb, sonicação está completa. Se não, continuar a sonicate. </li><li> Centrifugue as amostras sonicadas em 16100RCF durante 10 min a 4 ° C. Combine sobrenadantes, alíquotas em amostras de 1 ml e congelar-in Flash nitroge líquidon. Armazenar a -80 ° C. </li></ol> 6. CHIRP Hibridizar sondas de DNA biotiniladas para o RNA e isolar cromatina ligada. <ol><li> Tubos de descongelamento de cromatina à temperatura ambiente. </li><li> Preparar o tampão de hibridação (ver lista de buffer, prepare 2 ml por ml de cromatina). Vórtice para misturar. </li><li> Para uma amostra CHIRP típico usando 1 ml de lisado, remover 10 uL para RNA INPUT e 10 uL de ADN para INPUT e local em tubos de Eppendorf. Manter em gelo até posterior utilização. </li><li> Transferir 1 cromatina mL a 15 mL tubo Falcon. Adicionar 2 mL Tampão de Hibridização a cada tubo. Para o volume total &lt;1,5 ml, utilizar tubos Eppendorf. </li><li> Descongele sondas à temperatura ambiente. Nanodrop sondas para verificar quantidade se você não tê-lo usado em um longo tempo (100 sondas iM deve especificação ~ 500-600 ng / ul usando configuração DNA de cadeia simples). Adicionar volume apropriado de sondas específicas para os tubos (100 pmol por sonda 1 mL da cromatina, 1 uL de 100 sonda pmol / uL por 1 mL de cromatina).Misture bem. Incubar a 37 ° C durante 4 horas com agitação. </li><li> Com 20 min restante para a hibridação, preparar os grânulos C-1 magnéticos (armazenada a 4 ° C). Use de 100 L por 100 pmol de sondas. Lava-se com 1 mL Tampão de Lise não suplementado três vezes, utilizando a tira de íman DynaMag-2 a esferas separadas a partir de tampão. </li><li> Contas Ressuspender em volume original de tampão de lise e Suplemento com PMSF fresco, PI e Superase-in. Após 4 h de reacção de hibridação está completa, adicionar 100 grânulos uL a cada tubo. Misture bem. Incubar a 37 ° C durante 30 min com agitação. </li><li> Preparar tampão de lavagem (5 mL por amostra). Vórtice para misturar. Pré-aquecido a 37 ° C. Adicionar PMSF antes da utilização. </li><li> Lavar grânulos com 1 mL de tampão de lavagem por cinco vezes. Na primeira lavagem, utilizar-15 DynaMag tira magnética a esferas separadas, decantar, e ressuspender em 1 mL de tampão de lavagem. Transferir volume para tubo Eppendorf de 1,5 mL. Incubar a 37 ° C com agitação durante 5 min. </li><li> Em lavagens subseqüentes, girar cada tubo em um minicentrifuge, Definir amostra em DynaMag-2 tira magnética durante 1 min. Decantar amostra, seque os pingos com um Kimwipe, ressuspender em 1 ml de tampão de lavagem. Incubar a 37 ° C com agitação durante 5 min. Repetir para cinco lavagens totais. </li><li> Na última lavagem, ressuspender as esferas bem. Retirar 100 uL e reserve para o isolamento de RNA. Reserve uL 900 para fração de DNA. Coloque todos os tubos em DynaMag-2 fita magnética e remover o tampão de lavagem. Gire todos os tubos para baixo brevemente; colocá-los na faixa ímã. Retire o último pedaço de tampão de lavagem completamente com uma afiada ponta de pipeta 10 ul. </li></ol> 7. RNA Isolation Extrair fracção de ARN a partir de amostras Chirp para quantificar por qRT-PCR. <ol><li> Tome 100 l amostras de contas e 10 amostra de entrada uL RNA. Adicionam-se 85 uL RNA tampão pH 7,0 PK para o RNA INPUT. Contas Ressuspender em 95 uL de RNA PK pH 7,0. Adicionar 5 K Proteinease uL e incubar a 50 ° C durante 45 min com ponta a ponta de agitação. </li><li> Resumidamente girar todos os tubos eferver amostras durante 10 min em bloco de aquecimento a 95 ° C. </li><li> Arrefeça amostras em gelo, adicionar 500 uL TRIzol vórtice, vigorosamente, durante 10 seg. Incubar a temperatura ambiente durante 10 min. Conservar a -80 ° C ou vá para a etapa 4. </li><li> Adicionar 100 uL de clorofórmio para TRIzol amostras tratadas. Vortex vigorosamente durante 10 seg. Girar em 16100RCF numa centrífuga de bancada durante 15 min a 4 ° C. </li><li> Remover ~ 400 uL sobrenadante aquoso, evitando orgânica e de interface. </li><li> Adicionar 600 uL (1,5 de volume) de etanol a 100% e misturar bem. Centrifugue a amostra através MIRNeasy colunas mini. Lave 1x com ERP (MIRNeasy mini-kit), 2x por RPE com o protocolo do fabricante. Eluir com 30 uL sem nuclease H2O (NFH 2 O). </li><li> Tratar o produto de eluição com a DNA-RNA livre por protocolo do fabricante. Após a reacção está completa, aquecer a amostra durante 15 minutos a 65 ° C para inactivar qualquer completamente DNase restante. </li><li> Use 1 RNA isolado por microlitro bem para qRT-PCR para confirmar lncRNArecuperação. GAPDH é frequentemente utilizado como um controlo negativo. </li></ol> 8. Isolamento de DNA Extrair DNA de amostras de fração Chirp para identificar por seqüenciamento ou quantificar por qPCR. <ol><li> Preparar o tampão de eluição DNA (ver lista de buffer), 150 ul por amostra, incluindo entrada DNA. </li><li> Adicionar 1 0μL RNase A (10 mg / mL) e 10 uL de RNase H (10 U / ul) por ml de tampão de eluição de DNA, e vórtice para misturar. </li><li> Ressuspender cada amostra de grânulos em 150 uL de Tampão de Eluição com DNA RNases. (Ressuspender ENTRADA DNA em 140 uL) Incubar a 37 ° C durante 30 min com agitação. </li><li> Contas separadas e sobrenadante em DynaMag-2 tira magnética. Remover o sobrenadante e adicionar aos tubos etiquetados. </li><li> Prepara-se uma segunda alíquota de tampão de eluição de ADN com 10 uL de RNase A (10 mg / mL) e RNaseH (10 U / mL) exactamente como é feito em 8,2). Adicionar 150 uL de cada amostra (incluindo entrada do ADN), incubar e remover o sobrenadante. Colete todos sobrenadante (should ser ~ uL 300). </li><li> Adicionar 15 PK uL de cada amostra. Incubar a 50 ° C durante 45 min com agitação. </li><li> Pré-girar amarelo tubos fase de bloqueio de gel (5PRIME). Transferir as amostras de DNA a tubos de bloqueio de fase de gel, e adicionam-se 300 uL PhOH: Clorofórmio: isoamilo por amostra. Agita-se vigorosamente durante 10 min, e girar para baixo em uma centrífuga de bancada a 16100RCF durante 5 min a 4 ° C. Tome aquosa a partir do topo (~ uL 300). Adicionar 3 GlycoBlue uL, 30 NaOAc uL, e 900 uL de EtOH a 100%. Misture bem e armazenar a -20 ° C durante a noite. </li><li> Girar amostras a 16100RCF durante 30 min a 4 ° C. </li><li> Decante o sobrenadante cuidadosamente. Adicionar 1 mL de EtOH a 70% e vórtice para misturar. Girar em 16100RCF durante 5 min. Remover o sobrenadante com uma pipeta. Deixe secar por 1min. Ressuspender em 30 uL EB. </li><li> Amostras de DNA está pronto para análise por qPCR ou preparação de bibliotecas de alto rendimento de sequenciamento por protocolo Illumina. </li></ol> 10. Os resultados representativos A Figura 1 </strong&gt; descreve o fluxo de trabalho piar. Uma experiência CHIRP bem sucedida tipicamente enriquece RNA alvo significativamente ao longo do não-específicos RNAs. A Figura 2 mostra o enriquecimento de humano telomerase RNA (TERC) a partir de células HeLa sobre GAPDH, um RNA abundantes celulares que serve como um controlo negativo. Maioria dos RNAs TERC (~ 88%) presentes na célula foram puxados para baixo através da realização de CHIRP, enquanto que apenas 0,46% de GAPDH RNA foi recuperado, demonstrando um factor de enriquecimento de ~ 200 vezes. Sondas inespecíficas, tais como sondas de segmentação LacZ RNA, que não se expressa em células de mamíferos (Figura 2), pode ser usado como adicionais controlos negativos. Regiões de ADN que se espera que se ligam a lncRNA alvo são tipicamente enriquecida ao longo de regiões negativas, quando medido por qPCR. A Figura 3 mostra qPCR validação de quatro hotair ligados a sítios em fibroblastos de prepúcio humanos primários que determinados através da realização de CHIRP-seq na mesma linha celular, enquanto TERC e GAPDH DNA sítios siRVE como regiões de controlo negativo. Tanto o &quot;mesmo&quot; ea sonda &quot;estranha&quot; define rendeu enriquecimento comparável de espera hotair ligados sites sobre regiões negativas, marca registrada dos verdadeiros lncRNA sítios de ligação. Alta capacidade de seqüenciamento de DNA CHIRP enriquecido produz um mapa global de lncRNA sítios de ligação. A Drosophila lncRNA roX2 é conhecido para interagir com o cromossoma X de uma maneira que é necessária para a compensação de dosagem. A Figura 4 mostra roX2 perfil de ligação ao longo de um secção do cromossoma X. Tanto o &quot;mesmo&quot; e &quot;estranha&quot; as amostras já foram seqüenciados e seus ruídos originais foram eliminados para produzir uma faixa de sinais que se sobrepõem. Cada &quot;pico&quot; aqui indica um site de forte roX2 vinculativo. A faixa completa ea lista de roX2 genes alvo têm sido descritas na Chu et ai. 2011 17. <img alt="Figura 1." src="/files/ftp_upload/3912/3912fig1.jpg" /> Figura Fluxograma 1. Do procedi CHIRPDure. Cromatina é reticulado para lncRNA: aductos de proteína in vivo biotinilados sondas azulejos são hibridizados para alvejar lncRNA, e os complexos de cromatina são purificados utilizando esferas de estreptavidina magnéticos, seguido por lavagens rigorosas.. Nós eluir lncRNA DNA ligado ou proteínas com um coquetel de RNase A e H. A seqüência lncRNA suposta ligação está esquematizado na laranja. Anteriormente publicado em Chu et ai. 2011. 17 <img alt="Figura 2." src="/files/ftp_upload/3912/3912fig2.jpg" /> Enriquece a Figura 2. Chirp para o homem TERC RNA. TERC-asDNA sondas recuperar ~ 88% de RNA celular TERC e GAPDH indetectável. LacZ-asDNA sondas são utilizados como controles negativos e recuperar nem RNAs. A média + dp são mostrados. Anteriormente publicado em Chu et ai. 2011. 17 <img alt="Figura 3." src="/files/ftp_upload/3912/3912fig3.jpg" /> Figura 3. Hotair CHIRP-qPCR em humano primário parafibroblastos Eskin. NFKBIA, HOXD3-4, SERINC5 e ABCA2 são regiões que interagem com hotair. TERC e GAPDH serviram como controles negativos. A média + dp são mostrados. Anteriormente publicado em Chu et ai. 2011. 17 <img alt="Figura 4." src="/files/ftp_upload/3912/3912fig4.jpg" /> Figura 4. CHIRP-seq de dados de roX2 RNA em células de Drosophila SL2. &quot;Mesmo&quot; e &quot;estranho&quot; foram seqüenciados em separado; seus dados mesclar para refletir apenas picos comuns em ambos. A faixa fundida é mostrado. Anteriormente publicado em Chu et ai. 2011. 17

<ol>
	<li>Koziol, M. J., Rinn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+traffic+control+of+chromatin+complexes.">RNA traffic control of chromatin complexes.</a> Curr. Opin. Genet. Dev. 20, 142-148 (2010).</li><li>Mercer, T. R., Dinger, M. E., Mattick, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNAs:+insights+into+functions.">Long non-coding RNAs: insights into functions.</a> Nat. Rev. Genet. 10, 155-159 (2009).</li><li>Rinn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+demarcation+of+active+and+silent+chromatin+domains+in+human+HOX+loci+by+noncoding+RNAs.">Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs.</a> Cell. 129, 1311-1323 (2007).</li><li>Zhao, J., Sun, B. K., Erwin, J. A., Song, J. J., 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=Polycomb+proteins+targeted+by+a+short+repeat+RNA+to+the+mouse+X+chromosome.">Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome.</a> Science. 322, 750-756 (2008).</li><li>Kelley, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+spreading+of+the+Drosophila+dosage+compensation+complex+from+roX+RNA+genes+into+flanking+chromatin.">Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin.</a> Cell. 98, 513-522 (1999).</li><li>Pandey, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kcnq1ot1+antisense+noncoding+RNA+mediates+lineage-specific+transcriptional+silencing+through+chromatin-level+regulation.">Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation.</a> Mol. Cell. 32, 232-246 (2008).</li><li>Wang, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+long+noncoding+RNA+maintains+active+chromatin+to+coordinate+homeotic+gene+expression.">A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression.</a> Nature. 472, 120-124 (2011).</li><li>Khalil, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+human+large+intergenic+noncoding+RNAs+associate+with+chromatin-modifying+complexes+and+affect+gene+expression.">Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 11667-11672 (2009).</li><li>Zhao, J. <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+identification+of+polycomb-associated+RNAs+by+RIP-seq.">Genome-wide identification of polycomb-associated RNAs by RIP-seq.</a> Mol. Cell. 40, 939-953 (2010).</li><li>Meller, V. H., Wu, K. H., Roman, G., Kuroda, M. I., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=roX1+RNA+paints+the+X+chromosome+of+male+Drosophila+and+is+regulated+by+the+dosage+compensation+system.">roX1 RNA paints the X chromosome of male Drosophila and is regulated by the dosage compensation system.</a> Cell. 88, 445-457 (1997).</li><li>Franke, A., Baker, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rox1+and+rox2+RNAs+are+essential+components+of+the+compensasome,+which+mediates+dosage+compensation+in+Drosophila.">The rox1 and rox2 RNAs are essential components of the compensasome, which mediates dosage compensation in Drosophila.</a> Mol. Cell. 4, 117-122 (1999).</li><li>Gupta, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNA+HOTAIR+reprograms+chromatin+state+to+promote+cancer+metastasis.">Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis.</a> Nature. 464, 1071-1076 (2010).</li><li>Tsai, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+noncoding+RNA+as+modular+scaffold+of+histone+modification+complexes.">Long noncoding RNA as modular scaffold of histone modification complexes.</a> Science. 329, 689-693 (2010).</li><li>Zappulla, D. C., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+as+a+flexible+scaffold+for+proteins:+yeast+telomerase+and+beyond.">RNA as a flexible scaffold for proteins: yeast telomerase and beyond.</a> Cold Spring Harb. Symp. Quant. Biol.. 71, 217-224 (2006).</li><li>Nagano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Air+noncoding+RNA+epigenetically+silences+transcription+by+targeting+G9a+to+chromatin.">The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin.</a> Science. 322, 1717-1720 (2008).</li><li>Carter, D., Chakalova, L., Osborne, C. S., Dai, Y. F., Fraser, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+chromatin+regulatory+interactions+in+vivo.">Long-range chromatin regulatory interactions in vivo.</a> Nature. 32, 623-626 (2002).</li><li>Chu, C., Qu, K., Zhong, F. L., Artandi, S. E., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Maps+of+Long+Noncoding+RNA+Occupancy+Reveal+Principles+of+RNA-Chromatin+Interactions.">Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions.</a> Mol. Cell. , (2011).</li><li>Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+individual+mRNA+molecules+using+multiple+singly+labeled+probes.">Imaging individual mRNA molecules using multiple singly labeled probes.</a> Nat. Methods. 5, 877-879 (2008).</li></ol>

C. Chu e Chang HY são nomeados como inventores sobre um pedido de patente com base neste método.

Aqui descrevemos CHIRP-seq, um método de mapeamento em sites in vivo lncRNA ligação do genoma. Os principais parâmetros para o sucesso são as piscinas de divisão de azulejos sondas de oligonucleotídeos e reticulação glutaraldeído. A concepção de sondas de afinidade é simples, dada a sequência de RNA e não requer conhecimento prévio da estrutura do RNA ou domínios funcionais. O nosso sucesso com roX2, TERC, e hotair - três RNAs bastante diferentes em duas espécies - sugere que CHIRP-seq provavelmente generalizáveis ​​para lncRNAs muitos. Tal como com todas as experiências, os controlos de cuidados e adequada são necessários para interpretar os resultados. LncRNA diferente pode exigir titulação de condições, ea mudança criteriosa de condições, tais como seleção de sondas diferentes de afinidade ou reticuladores, pode realçar diferentes aspectos da RNA-cromatina interações. Como ChIP seguintes, nem todos os eventos de ligação são, necessariamente, funcional, e estudos adicionais são necessários para verificar as conseqüências biológicas da RNA occupancy em cromatina. No entanto, prevemos muitas aplicações interessantes desta tecnologia para os pesquisadores de outros cromatina associados lncRNAs, cujo número agora na casa dos milhares 8,9. Assim como ChIP-seq abriu a porta para a exploração do genoma de interações DNA-proteína, CHIRP-seq estudos do &quot;RNA interactoma&quot; pode revelar muitos novos caminhos da biologia.

Buffer List:
Dissolve a pellet of complete protease inhibitor in 1 ml water as 50x stock. Make 100 mM PMSF in isopropanol (100x stock). Superase-in is used as 200x stock. Store all at -20&deg;C.
Lysis Buffer:
50 mM Tris-Cl pH 7.0 
10 mM EDTA 
1% SDS 
Always add PMSF, P.I. and Superase-in fresh before use except when washing beads
Proteinase K Buffer (for DNA)
100 mM NaCl 
10 mM TrisCl pH 8.0 (For RNA use pH 7.0) 
1 mM EDTA 
0.5% SDS 
Add 5% by volume Proteainse K (Ambion AM2546 20 mg/ml) fresh before use
Hybridization Buffer
750 mM NaCl 
1% SDS 
50 mM Tris-Cl pH 7.0 
1 mM EDTA 
15% formamide (store in the dark at 4&deg;C) 
Always add PMSF, P.I. and Superase-in fresh before use
Wash Buffer
2x NaCl and Sodium citrate (SSC) (diluted from 20x SSC Invitrogen stock) 
0.5% SDS 
Always add PMSF fresh before use
DNA elution Buffer
50 mM NaHCO3 
1% SDS
Table of specific reagents and equipment:
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (EM grade)</td>
			<td>Sigma Aldrich</td>
			<td>G5882-10x10ml</td>
		</tr>
		<tr>
			<td>Motorized pellet mixer</td>
			<td>VWR</td>
			<td>V8185-904</td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Roche</td>
			<td>11873580001</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma Aldrich</td>
			<td>78830</td>
		</tr>
		<tr>
			<td>Superase-in</td>
			<td>Ambion</td>
			<td><a href="http://www.ambion.com/catalog/CatNum.php?AM2696">AM2696</a></td>
		</tr>
		<tr>
			<td>Bioruptor</td>
			<td>Diagenode</td>
			<td>UCD-200</td>
		</tr>
		<tr>
			<td>Falcon tubes (for sonication)</td>
			<td>Corning</td>
			<td>430790</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Ambion</td>
			<td>AM2546</td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28106</td>
		</tr>
		<tr>
			<td>C-1 magnetic beads</td>
			<td>Invitrogen</td>
			<td>65002</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma Aldrich</td>
			<td>P7626-25G</td>
		</tr>
		<tr>
			<td>DynaMag-15 magnet</td>
			<td>Invitrogen</td>
			<td>123-01D</td>
		</tr>
		<tr>
			<td>DynaMag-2 magnet</td>
			<td>Invitrogen</td>
			<td>123-21D</td>
		</tr>
		<tr>
			<td>MIRNeasy mini kit</td>
			<td>Qiagen</td>
			<td>217004</td>
		</tr>
		<tr>
			<td>Rnase H</td>
			<td>Epicentre</td>
			<td>R0601K</td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Sigma Aldrich</td>
			<td>R4875-100MG</td>
		</tr>
		<tr>
			<td>Phase Lock Gel Heavy</td>
			<td>5 PRIME</td>
			<td>2302810</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
		</tr>
		<tr>
			<td>Phenol:chloroform:Isoamyl</td>
			<td>Invitrogen</td>
			<td>15593-031</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Ricca</td>
			<td>RSOC0020-1C</td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>Ambion</td>
			<td>AM9515</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>J.T. Baker</td>
			<td>4057-06</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Invitrogen</td>
			<td>10010-049</td>
		</tr>
		<tr>
			<td>Elution Buffer (EB)</td>
			<td>Qiagen</td>
			<td>19086</td>
		</tr>
		<tr>
			<td>20x SSC</td>
			<td>Invitrogen</td>
			<td>15557-036</td>
		</tr>
		<tr>
			<td>10% SDS</td>
			<td>Invitrogen</td>
			<td>15553-027</td>
		</tr>
		<tr>
			<td>DNA-free</td>
			<td>Ambion</td>
			<td>AM1906</td>
		</tr>
		<tr>
			<td>Buffer kit</td>
			<td>Ambion</td>
			<td>AM9010</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Invitrogen</td>
			<td>15515-026</td>
		</tr>
	</tbody>
</table>

chromatin isolation by rna purification (chirp)

RNAs não-codificantes longos são os reguladores de cromatina principais estados para processos biológicos importantes, como a compensação de dosagem, imprinting e expressão de genes de desenvolvimento 1,2,3,4,5,6,7. A recente descoberta de milhares de lncRNAs em associação com os complexos de cromatina específicos de modificação, tal como o complexo Repressiva Polycomb 2 (PRC2) que medeia a histona H3 lisina 27 trimethylation (H3K27me3), sugere as funções gerais para lncRNAs numerosos estados na gestão de cromatina em um gene específico moda 8,9. Enquanto alguns lncRNAs são pensados ​​para trabalhar em cis em genes vizinhos, lncRNAs outros trabalhar em trans para regular genes localizados distantes. Por exemplo, Drosophila lncRNAs roX1 e roX2 regiões ligam numerosos no cromossomo X de células masculinas, e são críticas para a compensação de dosagem 10,11. No entanto, as localizações exatas dos seus locais de ligação não são conhecidos em alta resolução. Da mesma forma, humana lncRNA hotair pode afectar PRC2 ocupação em hundreds de genes do genoma 3,12,13, mas como especificidade o objectivo é claro. LncRNAs também pode servir como suportes modulares para recrutar o conjunto de complexos de proteínas múltiplas. O clássico trans-acting RNA andaime é o RNA TERC que serve como o modelo e andaime para a telomerase complexo 14; hotair também pode servir como um andaime para PRC2 e um desmetilase H3K4 complexo 13. Estudos anteriores de mapeamento de ocupação de RNA em cromatina revelaram percepções substanciais 15,16, mas apenas com um único locus do gene de cada vez. Os locais de ocupação da maioria dos lncRNAs não são conhecidos, e os papéis de lncRNAs na regulação da cromatina foram principalmente inferida a partir dos efeitos indiretos da perturbação lncRNA. Assim como imunoprecipitação de cromatina seguido por microarray ou seqüenciamento profunda (CHIP CHIP ou CHIP-seq, respectivamente) tem melhorado muito a nossa compreensão de interações DNA-proteína em escala genômica, aqui nós ilustrar um Recenqüentemente publicado estratégia para mapear ocupação RNA longo de todo o genoma em alta resolução 17. Este método de isolamento, cromatina pela RNA Purificação (CHIRP) (Figura 1), baseia-se na captura de afinidade de alvo lncRNA: Complexo de cromatina por ladrilhos anti-sentido-oligos, que gera então um mapa de sítios de ligação genómicos com uma resolução de várias centenas de bases com sensibilidade elevada e baixa do fundo. CHIRP é aplicável a muitos lncRNAs porque o desenho de afinidade sondas é simples, dada a sequência de RNA e não requer conhecimento da estrutura do RNA ou domínios funcionais.

Watch this Scientific Journal Video about Chromatin Isolation by RNA Purification ChIRP at JoVE.com

Chromatin Isolation by RNA Purification ChIRP

CHIRP é uma técnica recente e rápida de mapa genômico sítios de ligação de RNAs não-codificantes longos (lncRNAs). O método tira partido da especificidade dos oligonucleótidos anti-sentido ladrilhos para permitir que a enumeração de sítios lncRNA-bound genómicos.

Isolamento de cromatina por purificação de RNA (CHIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

chromatin-isolation-by-rna-purification-chirp

Research

JoVE Journal

Biology

Chromatin Isolation by RNA Purification (ChIRP)

85.7K visualizações.  Stanford University School of Medicine. CHIRP é uma técnica recente e rápida de mapa genômico sítios de ligação de RNAs não-codificantes longos (lncRNAs). O método tira partido da especificidade dos oligonucleótidos anti-sentido ladrilhos para permitir que a enumeração de sítios lncRNA-bound genómicos.

Vídeo: Chromatin Isolation by RNA Purification ChIRP

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Biologia

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolamento e purificação de Drosophila Neurônios periféricos por classificação Bead Magnética

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Geração de RNA / DNA híbrido em DNA genômico por transformação por meio de RNA contendo Oligonucleotídeos

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

Isolamento e Caracterização de RNA-Contendo exossomos

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

Endocitose em Fígado vivo seguidos de purificação de células do fígado por perfusão de fígado

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolamento e Purificação de cinesina partir Drosophila Os embriões

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Transcrição in vitro e nivelamento de Gaussia mRNA luciferase Seguido por HeLa transfecção celular

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Rápido isolamento e purificação de mitocôndrias para o transplante pela dissociação do tecido e Filtração Diferencial

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Geração e Purificação de INO80 cromatina remodelação Complexos Humanos e subcomplexos

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolamento de RNA Rato Pâncreas: Um tecido rico em Ribonuclease

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...