Os autores gostariam de agradecer a Michael Alifrangis e Ulla Abildtrup para a genotipagem de parasitas e Holm Christina de assistência técnica excelente. Este trabalho foi financiado pelo Howard Hughes Medical Institute (concessão 55.005.511), a Fundação Lundbeck (concessão R9-A840) e pela Fundação Niels Bohr.

1. Geração de recentemente selecionados eritrócitos infectados Para este ensaio, os melhores resultados são obtidos quando se utiliza as culturas de fresco seleccionados para a expressão de superfície da proteína PfEMP1. Nesta experiência particular, a P. 3D7 linhagem falciparum foi seleccionada utilizando anticorpos específicos como previamente descrito 1. Dia 1 <ol><li> Ul 200 de colheita de células do sangue compactados a partir de um p falciparum de cultura contendo IE fase final de 2-5% por centrifugação a 800 xg durante 8 minutos à temperatura ambiente. </li><li> Re-suspender o sedimento de sangue em 2 ml de 37 ° C uma solução de 0,75% de gelatina quente em um tubo de 14 ml estéril, e deixar durante 15-20 min a 37 ° C para permitir que o IE não infectados e anel estágio para sedimentar. </li><li> Colheita do IE de fase final, isto é, a fase superior, para um tubo novo ml 14 e lavar duas vezes com 10 ml de 37 ° C, lavagem quente RBC-media. </li><li> Diluir 50 ul de anticorpos específicos anti-PfEMP1 anticorpos em 2 ml de 37 ° C quentes RBC-lavagem e meios filtrantes estéreis. </li><li> Misture o IE de fase final lavada com anti-soros diluídos esterilizada num tubo de 14 ml estéril e incubar durante 30 min a 37 ° C com agitação suave. </li><li> Girar a 800 xg durante 8 minutos à temperatura ambiente, e lava-se duas vezes com 10 ml de 37 ° C, lavagem quente RBC-media. </li><li> Lave 50 ul da Proteína Dynabead A suspensão por duas vezes com RBC-lavagem mídia usando um ímã-15 DynaMaq. </li><li> Ressuspender as Dynabeads em 300 uL RBC-lavagem de mídia e adicioná-los ao IE fase final de lavada. Incubar durante 30 min a 37 ° C com agitação suave. </li><li> Transferir o tubo para o íman DynaMaq-15. Deixe por 1-2 minutos e retirar todo o líquido. </li><li> Ressuspender as Dynabeads em 4-5 ml de RBC-lavagem de mídia. Transfira a volta tubo sobre o ímã e deixe por 1-2 minutos antes de retirar todo o líquido. Repetir o passo de lavagem uma vez. </li><li> Transferir as pérolas lavadas para um 25 cm f cultura 2 estérillask contendo 200 ul de fresco glóbulos vermelhos. Adicionar 5-6 ml de Albumax mídia na cultura. Armazenar durante a noite a 5% de CO2 a 37 ° C. </li></ol> Dia 2 <ol start="12"><li> Remover as Dynabeads da cultura quando o IE seleccionou reocuparam as novas células vermelhas do sangue através da transferência de toda a cultura para um tubo de 14 ml estéril. Coloque o tubo sobre o ímã-15 DynaMaq e deixe por 1-2 min. Em seguida, despejar todo o líquido de volta para um frasco de cultura. </li><li> Cultura para o maior número possível de ciclos até a parasitemia de 5-10% e parasitas estão no ringstage. </li><li> IE devem ser analisadas por FACS para se certificar de que o fenótipo de superfície correcta, ou seja, a expressão da proteína de um ou outro and/orPF11_0008 PFD1235w foi obtida 1. </li></ol> 2. Preparação de Sondas As sondas de RNA anti-sentido foram obtidas a partir das regiões mais variáveis ​​do PFD1235w e o var PF11_0008 &lt;/ Em&gt; genes de P. falciparum 3D7 DNA genómico. O DNA foi amplificado por PCR e clonado no vector pSPT18 ou 19 para a transcrição, respectivamente de acordo com a descrição do fabricante (Roche). As sondas (580 pares de bases (pb) e 590 bp de comprimento) foram marcadas com digoxigenina (DIG) ou biotina utilizando um kit de marcação de ARN DIG ou a biotina RNA Mix rotulagem, respectivamente. Foi confirmada a especificidade das sondas tanto por análise de Northern blot e por um único rótulo análise FISH 1. 3. Smear fina e Fixação de Parasitas Antes de Hibridização In Situ Dia 1 Todos os passos são executados em um ambiente livre de RNase e todos os reagentes são RNase ou pré-tratadas com pirocarbonato de dietilo (DEPC) ou Zap RNase (Invitrogen). Os slides e lamínulas devem ser limpos com álcool para remover manchas de graxa fabricação residual. É importante realizar o protocolo, sem qualquer pausa entre os passosa fim de minimizar o risco de contaminação por nuclease. <ol><li> Fazer uma película fina de sangue pelo método de difusão padrão 3. Usando a lente 100x óleo objetivo do microscópio, contar o IE e calcular o percentual de IE na cultura. Verifique se os estágios do parasita estão em sincronia aproximada, no ringue e fases iniciais do ciclo de trofozoíto IE. Estes são pré-reprodução, uni-nucleadas fases, o mRNA do gene que expressa var e adequados para células individuais ensaios FISH transcrição deste tipo. </li><li> Transferir 50 uL da cultura parasita, a uma parasitemia de 5-10%, para um tubo Eppendorf de 1,5 ml sem RNase. Centrifugar durante 1 minuto a 2000 rpm à temperatura ambiente. </li><li> Remova a mídia e adicionar 50 pH PBS 7,2 em água tratada com DEPC. Agitar suavemente o tubo. Repetir o passo de sedimentação por centrifugação duas vezes para lavar as células livres a partir de meios de comunicação e os resíduos celulares. </li><li> Fazer quatro esfregaços finos de boa qualidade. Para cada esfregaço, fazer um esfregaço paraum diâmetro de 11 mm poço de uma corrediça 4 bem, ou seja, quatro lâminas de vidro, no total, em uma capa sem RNase (um passo crítico). Onda desliza brevemente ao ar para secar. Fazer três lâminas extra para slides negativo e um controlo para coloração única sonda para qualquer outro gene irrelevante usando uma sonda específica, uma outra corrediça para o tratamento com RNase e uma terceira lâmina contendo IE não expressa o gene de interesse. Deixe os slides para secar no banco por 10-20 min. </li><li> Fixar os filmes finos por adição de 60 ul de solução de fixação contendo paraformaldeído a 4% e 5% de ácido acético glacial para os poços. Deixe por 10 minutos no banco à temperatura ambiente. </li><li> Lavar as lâminas em 2x SSPE tampão durante 5 minutos por meio de agitação suave à temperatura ambiente. Resumidamente remover o excesso de líquido por tocar os poços que contêm as células suavemente com um lenço de papel. </li><li> Cobrir as lâminas com pepsina a 0,01% em HCl 0,01 M durante 2 min a 37 ° C (um passo extremamente crítico). Lavar as lâminas em 2x SSPE, durante 5 min à temperatura ambiente. </li><li> Dilui-se a solução de RNase para obter uma concentração final de 10 ug / ml. Cobrir os esfregaços de controlo negativo com 60 ul da solução de RNase. Incubar durante 30 min a 37 ° C e depois lavar as lâminas em 2x SSPE, durante 5 min à temperatura ambiente. </li><li> Prosseguir imediatamente para a etapa de hibridização. </li></ol> 4. A hibridação de sondas de RNA de lâminas fixas Dia 1 <ol><li> Preparar a câmara de hibridação, tal como um ThermoStar 100 HC4 ou uma caixa vazia ponta da pipeta colocar num forno limpo, por pulverização com RNase Zap e limpando-a limpa com um lenço de papel. Encher os canais da câmara de hibridação ou o fundo da caixa com água tratada com DEPC para certificar-se de que as lâminas não sequem durante a hibridação. Fechar a câmara e pré-aquecimento a 48 ° C. </li><li> Diluir as sondas em solução de hibridização para uma concentração final de 12 ng / ul. Desnaturar a solução de sonda a 65 ° C durante 5 minutos num bloco de aquecimento. Imediatamente relaxar no gelo. </li><li> Resumidamente as lâminas de ar seco após a lavagem 2x SSPE. Cuidadosamente remover o excesso de líquido em torno dos poços com um lenço de papel. </li><li> Abrir a câmara de hibridação e colocar as lâminas na câmara. Aplicar uma ou ambas as sondas em um volume total de 60 ul por cavidade. Cuidadosamente cobrir a gota de líquido com uma lamela de cobertura isenta de RNase usando fórceps estéreis. </li><li> Fechar a câmara e hibridizar as lâminas a 48 ° C durante pelo menos 16 horas durante a noite. </li></ol> 5. Conjugação de anticorpos e de amplificação de fluorescência Dia 2 Os passos que se seguem têm por base o TSA Plus Sistema Paleta de fluorescência a partir de Perkin Elmer. Todos os passos são realizados à temperatura ambiente. <ol><li> Lavar as lâminas três vezes em tampão TNT durante 5 minutos com agitação suave. </li><li> Bloquear os poços em 150 ul de tampão de TNB para 30 min. </li><li> Dilui-se o anticorpo conjugado com HRP α DIG-TNB em tampão na proporção de 1:100 e o anticorpo conjugado com HRP α-biotina em tampão de TNB, na proporção de 1:500. Remover qualquer excesso de tampão TNB se as lâminas com um lenço de papel. Ao detectar duas transcrições simultaneamente, ambos os anticorpos podem ir de uma vez. Aplicar uma quantidade total de 100 ul da solução de anticorpo-TNB por poço e cuidadosamente cobrir com uma lamela. Incubar as lâminas durante 2 horas. </li><li> Retire as lamínulas cuidadosamente. Lavar as lâminas em tampão TNT 3 vezes durante 5 min. </li><li> Dilui-se o fluoróforo FITC no reagente de amplificação tiramida em uma proporção de 1:500 e utilizar a 100 ul da solução em cada poço. Cobrir os poços com lamelas e incubar durante 12 min. </li><li> Remover a tampa desliza cuidadosamente e lavar as lâminas três vezes durante 5 min em tampão TNT, por agitação suave. </li><li> Dilui-se a Cyan3 fluoróforo no reagente de amplificação tiramida a uma razão de 1:500. Adicionam-se 100 ul porbem desta solução. Cobrir os poços com lamelas e incubar durante 12 min. </li><li> Remova a tampa desliza cuidadosamente e depois enxaguar as lâminas rapidamente por imersão em tampão TNT. </li><li> Lavar as lâminas três vezes com tampão TNT, durante 5 min. </li><li> Imergir os slides rapidamente em água tratada com DEPC. </li><li> Deixe as lâminas para o ar seco. Montar as lâminas com anti-fade reagente contendo DAPI. Selar os cantos das lamínulas com unha polonês. </li><li> Os slides estão prontos para microscopia. Manter as lâminas cobertas com folha de alumínio e armazenar a 4 ° C se a experiência é para ser completada no dia seguinte. A vedação completa dos lados das lâminas pode ser realizada 24 horas após a aplicação do reagente anti-desbotamento. Isto irá permitir que as lâminas a ser trabalhada por até uma semana após o protocolo ter sido completado. </li></ol> 6. A visualização das sondas hibridizadas <ol><li> Ligue o microscópio. Um microscópio de imunofluorescência padrão é suffictário para esse tipo de experiência, mas se você tiver acesso a um microscópio confocal você também pode usar isso para imagens 3D ou obter vídeos de suas células coradas. Permitir microscópio para aquecer por 15-30 min. Ligue o computador eo software. </li><li> Verificar a qualidade da coloração no microscópio de imunofluorescência. Escolha um local que contém alguns parasitas. </li><li> Ajustar o ganho de cada laser manualmente. Ajuste o pixel habitam a 4-6 m -1 s -1 e os passos para 512 x 512 pixels. </li><li> Tire uma foto de teste usando Frame Lambda. Reajustar o ganho do laser. </li><li> Ter um mínimo de 20 fotos boas de fluorescentes células individuais. Pelo menos 2-5 imagens de um campo que contém 5-20 parasitas são necessários a fim de obter uma visão apropriada a percentagem de parasitas positivos. </li></ol>

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RNA análise FISH, em contraste com os métodos tais como a Northern blotting e RT-PCR, permite a discriminação de transcritos de mRNA específicos ao nível da célula individual. Isto torna possível a discriminação entre as células transcricionalmente activos e inactivos, neste exemplo, P. falciparum protozoários parasitas dentro glóbulos vermelhos humanos. Tais células inteiras observações são muitas vezes necessário e pode desvendar importantes e romance padrões de transcrição 1. ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Ácido acético </td><td> Sigma / Aldrich </td><td> 338.826-100ml </td><td></td></tr><tr><td> Albumax mídia: solução RPMI Glutamina 1640 </td><td> Lonza </td><td> BE12-115F </td><td> 500 ml de RPMI 1640 Solução de 5 ml de glutamina </td></tr><tr><td> Albumax mídia: Gentamicina sulfato Albumax solução </td><td> Lonza </td><td> BE02-012E </td><td> 2,5 ml de gentamicina sulfato 50 ml solução Albumax- </td></tr><tr><td> Albumax solução: Hipoxantina </td><td> Sigma-Aldrich </td><td> H9377 </td><td> 0,8 g Hipoxantina </td></tr><tr><td> Albumax solução: AlbuMAX II </td><td> Eunvitrogen </td><td> 11021-037 </td><td> 200 g Albumax </td></tr><tr><td> Albumax solução: RPMI 1640 </td><td> Lonza </td><td> BE12-115F </td><td> 4 litros RPMI 1640 Dissolver com ímã no máximo. 50 ° C. Filtrar esterilizar e armazenar a -20 ° C em alíquotas. </td></tr><tr><td> Amberlite </td><td> Sigma </td><td> A5710-110G </td><td> Resina para deionize formamida. </td></tr><tr><td> Anti-biotina de cabra conjugado com peroxidase de pAb </td><td> Calbiochem </td><td> 203206 </td><td></td></tr><tr><td> Anti-Dig anticorpo </td><td> Novus biológicos </td><td> NB100-41330 </td><td></td></tr><tr><td> Anti-fade reagente com DAPI </td><td> Invitrogen </td><td> P36931 </td><td> Os meios de montagem tem a curar durante 24 h antes de selar completamente a lâmina. </td></tr><tr><td> Biotina RNA Labeling Mix </td><td&gt; Roche </td><td> 11685597910 </td><td></td></tr><tr><td> Câmera digital </td><td> Nikon vista digital DC-F11 </td><td></td><td></td></tr><tr><td> Lamelas </td><td> Menzel-glaser </td><td> 631-1570 </td><td></td></tr><tr><td> cultura em balão de 25 centímetros de superfície 2 Nunclon </td><td> Nunc </td><td> 156340 </td><td></td></tr><tr><td> DEPC </td><td> Fluka / Sigma </td><td> 32.490-100ml </td><td> 1 ml DEPC em 1000 ml de água deionizada. Adicionar uma barra de agitação e agita-se durante 12 h. Autoclave durante 30 minutos. </td></tr><tr><td> Câmera digital </td><td> Nikon vista digital DC-F11 </td><td></td><td></td></tr><tr><td> Dynabeads Proteína A </td><td> Invitrogen </td><td> 10002D </td><td></td></tr><tr><td> DynaMaq-15 Ímã </td><td> Invitrogen </td><td> 123-01D </td><td></td></tr><tr><td> Formamida 99% bioultra </td><td> Fluka/ Sigma </td><td> 47671-1l-F </td><td> Formamida desionizada: 5 g de resina de permuta iónica, por 100 ml de formamida. Agita-se 30 min. Filtrar através de papel Whatman. </td></tr><tr><td> Gelatina solução a 0 75%:. Gelatina </td><td> Sigma-Aldrich </td><td> G2500 </td><td> 3,75 g de gelatina em 500 ml de RPMI 1640. Aquece-se a 56 ° C para dissolver. Filtrar esterilizar quando 56 ° C. Armazenar a -20 ° C em alíquotas. </td></tr><tr><td> Gelatina solução a 0 75%:. RPMI 1640 </td><td> Lonza </td><td> BE12-115F </td><td> 3,75 g de gelatina em 500 ml de RPMI 1640. Aquece-se a 56 ° C para dissolver. Filtrar esterilizar quando 56 ° C. Armazenar a -20 ° C em alíquotas. </td></tr><tr><td> Solução de glutamina: L-glutamina </td><td> Sigma-Aldrich </td><td> G3126 </td><td> 14,6 g de glutamina L em 500 ml de NaCl a 0,9%. Dissolver, filtro sterilize e armazenar a -20 ° C em alíquotas. </td></tr><tr><td> Solução de glutamina: HCl </td><td> Sigma </td><td> H1758 </td><td> 14,6 g de glutamina L em 500 ml de NaCl a 0,9%. Dissolver, filtrar esterilizar e armazenar a -20 ° C em alíquotas. </td></tr><tr><td> Solução de hibridação: Formamida 99% Bio ultra-SSC 20x </td><td> Fluka / Sigma </td><td> 47671-1l-F </td><td> 20 ml Total, manter congelados a -20 ° C em alíquotas 10 ml de formamida desionizada </td></tr><tr><td> Solução de hibridação: concentrado 50x Denhardt </td><td> Sigma </td><td> D2532 </td><td> 20xSSC 5ml </td></tr><tr><td> Solução de hibridação: Yeast tRNARoche reagente de bloqueio </td><td> Sigma </td><td> R-6750 </td><td> 2 ml de 50x Denhardt 250 mg 20 ul por ml de ARNt de levedura </td></tr><tr><td><sTrong&gt; Solução de Hibridação: DNA de esperma de salmão </td><td> Fluka / Sigma </td><td> 31.149-106GF </td><td> 0,4 g Roche bloqueio reagente 1 ml de 10 mg por ml de DNA de esperma de salmão (Crítica: desnaturar spermDNA salmão a 96 ° C durante 5 min antes da adição à solução de hibridação) </td></tr><tr><td> Hybridizer ThermoStar 100 HC4 </td><td> Quantifoil Instruments GmbH </td><td> 1004-0011 </td><td> Pode ser substituído por forno de hibridação e câmaras de hibridização RNAse preenchidos com água DEPC. </td></tr><tr><td> Imersão em óleo UV transparente fluorescência livre </td><td> Sigma </td><td> 10.976-1EA </td><td></td></tr><tr><td> Microscópio de imunofluorescência ou confocal </td><td> Nikon D-Eclipse TE2000C </td><td></td><td></td></tr><tr><td> Nailpolish </td><td> Disponível em qualquer farmácia </td><td></td><td></td></tr><tr><td> PBS 20x: NaCl KCl Na 2 HPO 4 2H 2 O x KH 2 PO 4 DEPC H2O deionizada </td><td></td><td></td><td> Ajust pH para 7,4 160 g 4 g 23 g 4 g 1 l </td></tr><tr><td> Paraformaldeído a 4% </td><td> Fluka / Chemika </td><td> 76240 </td><td> 4 g PFA em 80 ml de PBS / DEPC. Aquece-se a 65 ° C até que o PFA dissolve. Adicionar 20 ml de PBS, permitir que a solução arrefeça. Ajustar o pH a 7,4. Filtrar. Armazenar em alíquotas a -20 ° C. </td></tr><tr><td> Paraformaldeído a 4% / ácido acético a 5% </td><td></td><td></td><td> 950 paraformaldhyde ul + 50 ul de ácido acético </td></tr><tr><td> Pepsina </td><td> Sigma / Aldrich </td><td> P7000 </td><td></td></tr><tr><td> Conjugado com peroxidase anti-biotina </td><td> Calbiochem </td><td> 203206 </td><td></td></tr><tr><td> RBC-lavagem de mídia: solução RPMI Glutamina 1640 </td><td> Lonza </td><td> BE12-115F </td><td> 500 ml de RPMI 1640 5 ml solução glutamina </td></tr><tr><td> RBC-lavagem de mídia: sulfato de gentamicina </td><td> Lonza </td><td> BE02-012E </td><td> 2,5 ml de gentamicina sulfato </td></tr><tr><td> RNase </td><td> Sigma / Aldrich </td><td></td><td> Faça um de 10 mg / ml de solução. </td></tr><tr><td> RNase livre tubo 1,5 ml </td><td> Ambion </td><td> AM12450 </td><td></td></tr><tr><td> RNase Zap </td><td> Ambion </td><td> AM9780 </td><td></td></tr><tr><td> Desliza 4 poços 11 milímetros </td><td> Thermo Scientific </td><td> MENZXER306W </td><td></td></tr><tr><td> SSC 20x: NaCl </td><td> Sigma / Aldrich </td><td> S9625 </td><td> NaCl 3 M (175 g / l), 0,3 M de citrato de Na 3 x H 2 O (88 g / l) Ajustar o pH a 7,0 com HCl 1M </td></tr><tr><td> SSC 20x: citrato de sódio desidratado </td><td> Sigma / Aldrich </td><td> W302600 </td><td> NaCl 3 M (175 g / l), 0,3 M de citrato de Na 3 x H 2 O (88 g / l) Ajustar o pH a 7,0 com HCl 1M </td></tr><tr><td> SSPE 20x: NaCl </td><td> Sigma / Aldrich </td><td> S9625 </td><td> 175,3 g de NaCl </td></tr><tr><td> SSPE 20x: NaH 2 PO 4 </td><td> Sigma / Aldrich </td><td> S0751 </td><td> 27,6 g NaH 2 PO 4. </td></tr><tr><td> SSPE 20x: 4 EDTA pó </td><td></td><td></td><td> 9,4 g de EDTA em pó Adicione a água DEPC, ajustar o pH 7,4. Automáticoclave durante 20 min </td></tr><tr><td> TNB tampão: tampão TNT </td><td></td><td></td><td> 10 ml de tampão TNT </td></tr><tr><td> TNB buffer: reagente de bloqueio </td><td></td><td></td><td> Reagente de bloqueio 0,05 g Para dissolver o reagente de bloqueio, aquecer a solução a 60 ° C durante uma hora com agitação. Armazenar a -20 ° C. (Derivado da Perkin Elmer TSA-protocolo). </td></tr><tr><td> TNT buffer: Tris / HCl </td><td> Sigma </td><td> T1503 </td><td> 1M Tris / HCl, pH 8,0 </td></tr><tr><td> Tampão TNT: NaCl </td><td> Sigma Aldrich </td><td> S9625 </td><td> 100 ml </td></tr><tr><td> TNT buffer: Tween20 </td><td> Sigma </td><td> 93773 </td><td> 5 M NaCl 30 ml 1 ml DEPC dH2O869 Ml Ajustar o pH para 7,5 à temperatura ambiente. (Derivado da Perkin Elmer TSA-protocolo). </td></tr><tr><td> TSA mais o sistema Cyanine3 / Fluoresceína </td><td> Perkin Elmer </td><td> NEL753000IKT </td><td> Leia o protocolo da TSA Plus Sistema Paleta de fluorescência cuidadosamente antes de iniciar o experimento. </td></tr><tr><td> Tubos de 14 ml estéril </td><td> Almeco - CM LAB Aps </td><td> 91016 </td><td></td></tr></tbody></table>

analysis of single-cell gene transcription by rna fluorescent in situ hybridization (fish)

Adesão de eritrócitos infectados por Plasmodium falciparum (IE) para receptores endoteliais humanas durante infecções de malária é mediada pela expressão de PfEMP1 variantes proteicas codificadas pelos genes var. O P. haplóide falciparum genoma alberga aproximadamente 60 diferentes genes var de que um só tenha sido acreditados para ser transcrito por célula de cada vez, durante o estágio de sangue da infecção. Como regra, tais mutuamente exclusivos da transcrição de genes var é alcançado não é clara, assim como a identificação de genes individuais var ou sub-grupos de genes var associados com diferentes receptores e da consequência da ligação diferencial sobre os resultados clínicos de P. falciparum. Recentemente, o paradigma transcrição mutuamente exclusivos tem sido posta em dúvida por ensaios de transcrição com base no P. indivíduo falciparum identificação transcrição em inf únicoected células eritrocitários usando RNA hibridização in situ fluorescente (FISH) de análise de transcrição do gene var pelo parasita em núcleos individuais de P. falciparum IE 1. A seguir, apresentamos um protocolo detalhado para a realização do método RNA-FISH para análise da transcrição de genes var em núcleos única de P. falciparum infectados eritrócitos humanos. O método baseia-se na utilização de digoxigenina e biotina marcado com sondas de RNA anti-sentido, utilizando o sistema de Fluorescência de TSA Além paleta 2 (Perkin Elmer), as análises microscópicas e P. recém seleccionada falciparum IE. O método de hibridização in situ pode ser usado para controlar a transcrição e a regulação de uma variedade de genes expressos durante as diferentes fases do P. falciparum ciclo de vida e é adaptável a espécie de malária outros parasitas e outros organismos e tipos de células.

A Figura 1 ilustra um fluxograma dos passos principais e do tempo de duração da metodologia FISH RNA. Uma série de imagens representativas de bem e mal conservadas experiências FISH manchadas de mRNA utilizando P. única falciparum IE são mostrados na Figura 2. Este protozoário intracelular tem um núcleo (1-1,5 um de diâmetro) de pequeno porte, que é tingido de azul com DAPI. Vinte e quatro horas após a invasão dos eritrócitos e...

Watch this Scientific Journal Video about Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH at JoVE.com

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

Fluorescente<em&gt; In situ</em&gt; Hibridização (FISH) para identificar transcritos de ARNm em células individuais permite a análise da actividade poligênica tais como a transcrição simultânea de mais do que um membro da<em&gt; Var</em&gt; Família multigênica em<em&gt; Plasmodium falciparum</em&gt; Eritrócitos infectados<sup&gt; 1</sup&gt;. A técnica é adaptável e pode ser usado em diferentes tipos de genes, células e organismos.

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

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

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Research

JoVE Journal

Biology

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

15.4K visualizações.  University of Copenhagen. Fluorescente In situ Hibridização (FISH) para identificar transcritos de ARNm em células individuais permite a análise da actividade poligênica tais como a transcrição simultânea de mais do que um membro da Var Família multigênica em Plasmodium falciparum Eritrócitos infectados 1. A técnica é adaptável e pode ser usado em diferentes tipos de genes, células e organismos.

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafish Whole Mount Alta Resolução Duplo Fluorescente In Situ Hibridização

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

Biologia

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

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

Fluorescence in situ hybridization FISH Protocol in Human Sperm

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

Fluorescência In situ Hybridization (FISH) Protocolo no esperma humano

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

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 ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Radioativo In situ Hibridação para detecção de padrões de expressão dos genes nos tecidos diversos

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

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

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

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

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

Detecção de RNA viral por fluorescência<em&gt; In situ</em&gt; Hybridization (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

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

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

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

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

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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

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

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; Em situ</em&gt; Hibridação em embriões de Mount inteiros e histologia de células Adaptado para Elasmobrânquios Marinhos

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

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

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

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

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

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

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

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Análise da Global RNA Síntese no nível da célula única seguinte hipóxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...