Este estudo foi apoiado por fundos de Rita Allen doação da Fundação para Seena Ajit. Os autores gostariam de agradecer Erika Balogh e Dr. Soumitra Ghoshroy da Universidade da Carolina do Sul Centro de Microscopia Eletrônica para o uso do instrumento, assistência técnica e científica.

Todos os experimentos com amostras de sangue de humanos e roedores foram executados em conformidade com todas as diretrizes, regulamentos e agências reguladoras. Seres humanos foram registrados após o consentimento informado aprovado pela Drexel University College of Medicine Institutional Review Board e todos os procedimentos de estudos realizados com animais foram aprovados pelo Institutional Animal Care e do Comitê Use Drexel. 1. Purificação exossomo de sangue (~ 5,5 h) DICAS <ul><li class="jove_content"> Nós ressuspender exossomos a partir de um tubo de sangue (~ 10 ml de sangue humano e ~ 2 ml de sangue mouse) em 300 mL de tampão de lise RNA a partir do kit de isolamento miRNA miRvana. </li><li class="jove_content"> Para purificar exosomes de meios de cultura de células, o meio é centrifugado a 500 xg durante 10 min para remover as células e também filtrada através de um filtro de 0,22 um, após centrifugação a 12.000 x g. A purificação exosome de meios de cultura celularnão necessita de diluição em PBS, PBS ou a solução de lavagem do segundo passo de ultracentrifugação. </li><li class="jove_content"> Se forem utilizados os exosomes purificadas para estudos de proteómica, a adição de um passo de centrifugação em gradiente de sacarose após o passo de ultracentrifugação vai diminuir a quantidade de agregados de proteína e contaminantes 15. </li><li class="jove_content"> A morfologia observada de exosomes pode variar de acordo com a fonte e as etapas de processamento para a ETM. Não observamos a morfologia em forma de copo para exossomos, que foi anteriormente atribuído a diferenças no processamento da amostra 9,15. Nossas imagens TEM, incluindo a rotulagem immunogold são semelhantes aos relatos anteriores de diferentes fontes, incluindo sangue, biliar e cultura cortical derivado exosomes 9,16,17. </li></ul><ol><li> Inverter EDTA tubo de revestimento que contém o sangue de 5x e coloque na posição vertical à temperatura ambiente por 10-60 min. </li><li> Centrifugar a 2.000 xg durante 15 min a 4 ° C. </li&gt; <li> Recolher a fase superior, o plasma, em tubos de 15 ml e guardar em gelo. </li><li> Dilui-se fluido com um volume igual de PBS. Centrifugar 30 min a 2000 xg, a 4 ° C. </li><li> Transferir para tubos de centrífuga, adicionar 1X PBS, para um volume total de 24 ml e centrifugar 45 min a 12000 xg, a 4 ° C. </li><li> Transfira para os tubos de ultracentrifugação e centrifugar 2 horas a 110.000 xg, a 4 ° C. </li><li> Ressuspender o pellet em PBS e centrifugar 1 hora a 100.000 x g a 4 ° C. </li><li> Ressuspender o sedimento em tampão apropriado (tampão de lise de ARN). </li><li> Ressuspender em PBS ou tampão RIPA para microscopia eletrônica e análise de western blot, respectivamente. </li></ol> 2. Purificação de RNA usando um kit de isolamento miRvana miRNA modificação (~ 1 hora) DICAS <ul><li class="jove_content"> Preparar a área para isolar RNA, usando equipamentos de proteção individual (EPI), limpando superfícies e pipetas com RNase Zap, e usando pontas com filtro estéreis e tubos. </li><liclass = &quot;jove_content&quot;&gt; Embora o protocolo do fornecedor dá a opção de purificação só miRNA, sempre isolar RNA totais, incluindo mRNA e miRNA para que possamos estudar tanto a partir da mesma amostra. </li><li class="jove_content"> Adicionando um passo de tratamento de DNase na coluna remove vestígios de ADN que podem estar presentes. Dependendo da fonte de exosomes, o DNA foi relatado que estavam ausentes em exosomes purificadas a partir de algumas fontes, incluindo o rato e as linhas de mastócitos humanos 8 mas as sequências de ADN cromossómico foram detectados em exosomes purificadas a partir de meios de cultura de cardiomiócitos 18. </li><li class="jove_content"> Nós reduziu o volume de solução de lavagem de 1-350 ul (Passo 2.4), porque o fornecedor disponibiliza apenas suficiente uma solução de lavagem para uma adição de 700 ul. Se o volume não seja reduzido, não há solução suficiente deixada para utilizar todo o conteúdo do kit. Demais etapas foram realizadas de acordo com as recomendações do fabricante. </li></ul><ol><li> Adicione 1/10 volume de aditivo miRNA homogeneizado (30 uL) e incubar em gelo durante 10 min. </li><li> Extrai-se com um volume de ácido-fenol: clorofórmio, igual ao volume de lisado inicial. Centrifugar e recuperar a fase superior num novo tubo. </li><li> Adicionar 1,25 volumes de etanol a 100% (375 ul). Vortex. Aplicar para filtro de cartucho e centrifugar 15 segundos a 10.000 x g. </li><li> Adicione metade do volume de miRNA solução de lavagem recomendado 1 (350 mL) e centrifugar 15 segundos a 10.000 x g. </li><li> Adicionar 10 ul de DNase 1 a 70 ul de tampão de RDD (Qiagen) para cada amostra. Adicionar 80 ul para a coluna e incubar 15 min temperatura ambiente. </li><li> Adicionar uma solução de lavagem (350 ul) e de centrifugação de 15 segundos a 10.000 x g. </li><li> Adicionar solução de lavagem de 2/3 (500 mL) e centrifugar 15 s a 10.000 x g. REPEAT. </li><li> Eluir com 40 ul de 95 ° C sem nuclease H2O </li><li> Determinar a concentração de ARN e diluir o ARN a 100 ng / mL. </li></ol> 3. miRNA Profiling de Exosomes de Blood Usando Taqman Low Density Array (TLDA) Cartões (~ 6,5 hr) DICAS <ul><li class="jove_content"> Preparar a área para isolar RNA, vestindo PPE, limpando superfícies e pipetas com RNase Zap, e usando pontas com filtro estéreis e tubos. </li><li class="jove_content"> Pré-amplificação passo (pré-amplificador) é recomendado para 1-350 ng de RNA (350-1,000 amostras ng não requerem passo de pré-amplificador, após a síntese de cDNA). É importante determinar se o pré-amplificador é necessário para a sua amostra porque você deve tratar todas as amostras em condições de igualdade. Passo para pré-amplificador adiciona custo e os valores de Ct mais baixo tem um cut off aceitável (32 em vez de 40). </li><li class="jove_content"> É útil para preparar a reacção de cDNA em tubos em vez de uma tira de chapa de acordo com o número de cartas que pode ser executado num único dia. Isto irá minimizar a congelação-descongelação de amostras de cDNA que não serão processados ​​no mesmo dia. </li><li class="jove_content"> Exossomos provenientes de diferentes fontes têm difealugar quantidades de RNA. Este protocolo permite que um máximo de 3 ul de molde de ARN para a reacção de síntese de cDNA. Dependendo da fonte, a quantidade de RNA exosomal que pode ser isolado talvez limitada. Enquanto um tecido típico renderia o suficiente RNA que cDNA seria feito a partir de 100 ng de RNA em 3 mL de volume, exossomos de sangue humano ou de meios de cultura de células humanas, muitas vezes contêm menos RNA e cDNA deve ser feita a partir de material menos de partida. Rato exosomes sangue contêm níveis mais elevados de RNA e de pelo menos 100 ng em 3 ul pode ser obtida. </li></ul><ol><li> Para preparar cDNA a partir de RNA usando Pools Megaplex purificada primers da transcriptase reversa (Grupo A e Grupo B contêm primers para os miRNAs em cartões microfluídicos matriz A e B), descongelar os componentes de reação no gelo. </li><li> Rótulo dois tubos de 1,5 ml RNase para reação de RT com tanque primers ou Grupo B primários e adicionar componentes a fim gráfico. Adicionar pelo menos um volume adicional de componentes de dois ou mais reacções ou fazer uma mistura principal, tal como recomendado por tele fornecedor. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Componentes da reacção RT </td><td> Concentração Componente </td><td> Volume de uma amostra (total V / amostra = 4,5 mL) </td></tr><tr><td> Água livre de nuclease </td><td></td><td> 0,20 mL </td></tr><tr><td> Megaplex tampão RT (10X) </td><td> 1X </td><td> 0,80 mL </td></tr><tr><td> dNTPs (100 mM) </td><td> 4,4 mM </td><td> 0,20 mL </td></tr><tr><td> MgCl2 (25 mM) </td><td> 5mM </td><td> 0,90 mL </td></tr><tr><td> Inibidor de RNase (20U/μl) </td><td> 2 U </td><td> 0,10 mL </td></tr><tr><td> Megaplex RT Primers A ou B (10X) </td><td> 1X </td><td> 0,80 mL </td></tr><tr><td> MultiScribe transcriptase reversa </td><td> 75 U </td><td> 1,50 mL </td></tr></tbody></table><ol start="3"><li> Inverter tubos de 6 vezes e centorifuge brevemente. Em seguida, pipeta de 4,5 ul da mistura de reacção de RT em 8 MicroAmp Tiras de tubo ou uma placa de reacção óptica de 96 poços MicroAmp. </li><li> Adicionam-se 100 ng de RNA e ajustar o volume para 3 mL de água tratada com DEPC ou adicionar 3 ul de RNA total, misture com a ponta da pipeta, e fechar os tubos ou de placas. Girar rapidamente e incubar no gelo por 5 min. </li><li> Carregar a reacção para dentro da máquina de PCR e executá-la sob as seguintes condições (como recomendado pelo protocolo da Applied Biosystems Megaplex Piscinas): </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Etapa </td><td> Temp </td><td> Tempo </td></tr><tr><td> Ciclo (40x) </td><td> 16 ° C </td><td> 2 min </td></tr><tr><td></td><td> 42 ° C </td><td> 1 min </td></tr><tr><td></td><td> 50 ° C </td><td> 1 seg </td></tr><tr><td> Manter </td><td> 85 ° C </td><td> 5 min </td></tr><tr><td> Manter </td><td> 4 ° C, </td><td> ∞ </td></tr></tbody></table><ol start="6"><li> Armazenar de cDNA a -20 ° C (boa durante pelo menos uma semana), ou continuar a etapa de pré-amplificação que podem ser armazenadas durante uma semana. </li><li> Descongele primers pré-amplificador para o Grupo A e Grupo B no gelo e inverter para misturar. Swirl Taqman Pré-Amp mestre misturar e manter em gelo também. </li><li> Seguir o gráfico de adição de pelo menos um volume adicional de componentes de dois ou mais reacções ou fazer uma mistura principal, tal como recomendado pelo fornecedor. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Componente Reação Pré-Amp </td><td> Volume de uma amostra </td></tr><tr><td> Água livre de nuclease </td><td> 7,5 mL </td></tr><tr><td> Taqman Mix Master PreAmp (2X) </td><td> 12,5 mL </td></tr><tr><td> Primers pré-amplificador Megaplex A ou B (10x) </td><td> 2,5 mL </td></tr></tbody></table><ol start="9"><li> Pipetar 2,5 mL RT product MicroAmp em tiras de 8-tubo ou uma placa de reacção óptica de 96 poços e dispensar MicroAmp 22,5 ul da mistura de reacção PreAmp para cada poço. </li><li> Selar a placa, incubar no gelo 5 min e em seguida, coloque a amostra na máquina de PCR. Executar sob as seguintes condições. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Etapa </td><td> Temp </td><td> Tempo </td></tr><tr><td> Manter </td><td> 95 ° C </td><td> 10 min </td></tr><tr><td> Manter </td><td> 55 ° C </td><td> 2 min </td></tr><tr><td> Manter </td><td> 72 ° C </td><td> 2 min </td></tr><tr><td> Ciclo (12x) </td><td> 95 ° C </td><td> 15 seg </td></tr><tr><td></td><td> 60 ° C </td><td> 4 min </td></tr><tr><td> Manter </td><td> 99,9 ° C </td><td> 10 min </td></tr><tr><td> Manter </td><td> 4 ° C, </td><td> ∞ </td></tr></tbody></table><ol start="11"><li> Após a reacção do pré-amplificador é terminado, centrifugar brevemente os tubos ou as placas e, em seguida, adicionar 75 ul de 0,1 x TE pH 8,0 a cada poço ou tubo. </li><li> Seal e misture e girar rapidamente. Armazenar o produto diluído durante uma semana a -20 ° C, ou continuar a carregar a placa. </li><li> Combinar o seguinte num tubo de 1,5 ml RNase em gelo. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Componente </td><td> Volume de um cartão </td></tr><tr><td> TaqMan Universal PCR Master Mix, No AmpErase UNG (2X) </td><td> 450 mL </td></tr><tr><td> Produto pré-amplificador diluído </td><td> 9 mL </td></tr><tr><td> Água livre de nuclease </td><td> 441 mL </td></tr></tbody></table><ol start="14"><li> Inverter o tubo de misturar e centrifugar brevemente. Pipetar 100 ul da mistura de reacção de PCR em cada porta do cartão de matriz MicroRNA Taqman. </li><li> Centrifugar o cartão de 2 x 1.000 xg por 1 min. Selar a placa e executá-lo usando o modelo fornecido com os primers no manual da Applied Biosystems e os Taqman configurações de matriz densidade 384 bem baixa. Usamos Applied Biosystems 7900HT Rápido Real-Time PCR System. </li></ol> 4. Análise por ARNm Taqman (~ 1,5 h) <ol><li> Para preparar a reacção de qPCR, misturar os componentes da reacção, no fim do gráfico. Todas as amostras serão analisados ​​com sondas de iniciadores para o gene de interesse, bem como rRNA 18S como normalizador do gene. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Reacção de componentes Taqman </td><td> Volume de uma amostra </td></tr><tr><td> Água livre de nuclease </td><td> 7 ul (ajuste com base na amostra individual) </td></tr><tr><td> Taqman rápido Master Mix (2X) </td><td> 10 ul </td></tr><tr><td> Sondas Taqman iniciadores (20x) </td><td> 1 ul </td></tr><tr><td> cDNA a partir de 100 ng de ARN </td><td> 2 &amp; mu, L (ajuste com base na amostra individual) </td></tr></tbody></table><ol start="2"><li> Executar a placa de 96 poços utilizando o bloco de 96 poços rápida, tal como recomendado pelo fabricante (Applied Biosystems 7900HT Rápido Real-Time PCR System). </li></ol>

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Neste protocolo, vamos mostrar a quantificação dos miRNAs e mRNAs de exossomos purificados por centrifugação diferencial dos meios de sangue e cultura. Exosomes têm diversos componentes que dependem da sua origem e estão envolvidos em várias funções biológicas, incluindo a resposta imunológica, a apresentação de antigénio, a comunicação intracelular, bem como a transferência de RNA e proteínas 9,11,12,19,20. Embora a forma e tamanho é um determinante de pureza exossomo, uma série de artigo...

MiRNAs noncoding Curtas modular a expressão do gene por ligação ao ARNm alvo. Semente sequência complementaridade de pares de bases ~ 7 permite miRNA se liguem ao ARNm alvo, resultando na inibição da tradução ou na redução na estabilidade do mRNA, o que pode resultar na diminuição da expressão da proteína alvo 1. Pesquisas na última década tem se mostrado inequivocamente um papel fundamental para miRNAs em mediar funções celulares. Também tem havido considerável esforço dirigido para dissecar miRNA alterações moleculares subjacentes a diversas doenças mediadas por 2,3. Além disso, a recente identificação de miRNAs estáveis ​​em fluidos corporais 4-6 pavimentou o caminho para a sua utilização como novos biomarcadores passíveis de diagnóstico clínico. Um modo de transporte miRNA em fluidos corporais é via exossomos, pequenas vesículas que transportam mRNAs, proteínas, mediadores lipídicos e miRNAs em células receptoras via cir arterial sistêmicaion 7-14. Isto resulta na modulação da expressão de genes em células do receptor e representa um novo mecanismo de comunicação celular. Por exemplo, as células são capazes de modular processos de imuno-regulação através da secreção e / ou absorventes exosomes contendo biomoléculas envolvidas na inflamação, tais como a interleucina-1β (IL1β), factor de necrose tumoral α (TNF), factor de crescimento transformante-β5 (TGFβ5) e os miRNAs que regulam estes genes 13. Como expressão de miRNA aberrante é uma característica comum em uma variedade de doenças humanas, estas moléculas oferecem novas oportunidades para a descoberta e validação de novos alvos terapêuticos 2. Circulating miRNAs estão presentes em todos os fluidos corporais e é sabido que a composição do exosomes é diferente baseada nas células de origem a partir do qual eles foram libertados. Assim, eles oferecem um caminho para estudar o estado fisiológico das células e como as células alteram a sinalização atéts em resposta ao estresse, incluindo doenças. Estudar as alterações na composição exosome pode fornecer insights sobre a transdução de sinal e investigar a sua utilidade potencial como biomarcadores ou rotas de intervenção terapêutica. Aqui vamos demonstrar a purificação de exossomos a partir de várias fontes baseadas em protocolos publicados. Estes exosomes será usado para o isolamento de ARN seguida por qPCR para identificar e medir os níveis de miRNAs presentes nas exosomes.

<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>EDTA coated vacutainer (10 ml tubes)</td>
 <td>BD Diagnostics</td>
 <td>366643</td>
 <td>For exosome purification from human blood</td>
 </tr>
 <tr>
 <td>EDTA coated vacutainer (2 ml tubes)</td>
 <td>BD Diagnostics</td>
 <td>367841</td>
 <td>For exosome purification from mouse blood</td>
 </tr>
 <tr>
 <td>PAXgene Blood RNA Tube</td>
 <td>BD Diagnostics</td>
 <td>762165</td>
 <td>For miRNA isolation from total blood</td>
 </tr>
 <tr>
 <td>miRVana microRNA isolation kit</td>
 <td>Ambion</td>
 <td>AM1561</td>
 <td></td>
 </tr>
 <tr>
 <td>Acid-Phenol: CHCl3</td>
 <td>Ambion</td>
 <td>9721G</td>
 <td></td>
 </tr>
 <tr>
 <td>DNase 1</td>
 <td>Qiagen</td>
 <td>79254</td>
 <td></td>
 </tr>
 <tr>
 <td>TaqMan Universal PCR Master Mix, No AmpErase UNG</td>
 <td>Applied Biosystems</td>
 <td>4326614</td>
 <td>For TLDA cards</td>
 </tr>
 <tr>
 <td>Megaplex RT rodent Pool Set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444746</td>
 <td></td>
 </tr>
 <tr>
 <td>Megaplex preamp rodent Pool set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444747</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman Array Rodent MicroRNA A+B set V3.0</td>
 <td>Applied Biosystems</td>
 <td>4444909</td>
 <td></td>
 </tr>
 <tr>
 <td>Megaplex RT Human Pool set V3.0</td>
 <td>Applied Biosystems</td>
 <td>4444745</td>
 <td></td>
 </tr>
 <tr>
 <td>Megaplex Preamp human pool set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444748</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman Array Human MicroRNA A+B Cards Set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444913</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman MicroRNA RT kit</td>
 <td>Applied Biosystems</td>
 <td>4366596</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman fast universal PCR master mix </td>
 <td>Applied Biosystems</td>
 <td>4366072</td>
 <td>For mRNA qRT-PCR</td>
 </tr>
 <tr>
 <td>Tumor necrosis factor (primer probe)</td>
 <td>Applied Biosystems</td>
 <td>Hs01113624_g1 </td>
 <td></td>
 </tr>
 <tr>
 <td>Vascular endothelial growth factor A (primer probe)</td>
 <td>Applied Biosystems</td>
 <td>Hs00900055_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>Maxima First Strand cDNA Synthesis Kit for RT-qPCR</td>
 <td>Thermo Scientific</td>
 <td>K1642</td>
 <td>For mRNA</td>
 </tr>
 <tr>
 <td>HSP70 antibody</td>
 <td>Abcam</td>
 <td>ab94368 </td>
 <td>For western blot</td>
 </tr>
 <tr>
 <td>Anti-rabbit IgG-Gold</td>
 <td>Sigma</td>
 <td>G7402</td>
 <td>For electron microscopy</td>
 </tr>
 <tr>
 <td>Rabbit-anti CD81</td>
 <td>Sigma</td>
 <td>SAB3500454</td>
 <td></td>
 </tr>
 <tr>
 <td>Nickel 300 mesh carbon formvar grids</td>
 <td>Electron Microscopy Sciences</td>
 <td>FCF300-Ni</td>
 <td></td>
 </tr>
 <tr>
 <td>Copper 300 mesh carbon formvar grids</td>
 <td>Electron Microscopy Sciences</td>
 <td>FCF300-Cu</td>
 <td></td>
 </tr>
 <tr>
<td></td>
<td></td>
<td></td>
<td>Table 1. Table of specific reagents.</td>
</tr>
 </tbody>
 </table>

purification and microrna profiling of exosomes derived from blood and culture media

MiRNAs estáveis ​​estão presentes em todos os fluidos do corpo e em alguns miRNAs circulantes são protegidos da degradação por sequestração em pequenas vesículas chamadas exosomes. Exosomes podem fundir com a membrana plasmática que resulta na transferência de RNA e proteínas da célula-alvo. As suas funções biológicas incluem resposta imune, a apresentação de antigénio, e a comunicação intracelular. Entrega de miRNAs que podem regular a expressão do gene em células receptoras através do sangue, abriu novas possibilidades para intervenção alvo. Além de oferecer uma estratégia para a entrega de medicamentos ou agentes terapêuticos RNA, o conteúdo exosomal podem servir como biomarcadores que podem ajudar no diagnóstico, determinar as opções de tratamento e prognóstico. Aqui vamos descrever o procedimento para analisar quantitativamente miRNAs e RNAs mensageiro (mRNA) de exossomos secretados no sangue e meio de cultura de células. Purificados exosomes será caracterizado através da análise de western blot para exosmarcadores Omal e PCR para mRNAs de interesse. Microscopia eletrônica de transmissão (TEM) e immunogold rotulagem será usado para validar exosomal morfologia e integridade. O RNA total será purificada a partir destes exosomes para assegurar que podemos estudar tanto mRNA e miRNA partir da mesma amostra. Depois de validar a integridade do RNA por Bioanalyzer, vamos realizar um meio de transferência quantitativa PCR em tempo real (qPCR) para identificar o miRNA exosomal usando Taqman Matriz de Baixa Densidade (TLDA) cartas e estudos de expressão gênica de transcrições de interesse. Estes protocolos podem ser utilizados para quantificar as alterações na miRNAs exosomal em pacientes, modelos de roedor e meios de cultura de células antes e após a intervenção farmacológica. Conteúdo exosomal variar devido à fonte de origem e as condições fisiológicas de células que secretam exossomos. Essas variações podem fornecer uma visão sobre como as células e os sistemas de lidar com o estresse ou perturbações fisiológicas. Nossos dados representativos mostrar variations em miRNAs apresentam em exossomos purificados a partir do sangue mouse, sangue humano e os meios de cultura de células humanas. Aqui vamos descrever o procedimento para analisar quantitativamente miRNAs e RNAs mensageiro (mRNA) de exossomos secretados no sangue e meio de cultura de células. Purificados exosomes será caracterizado através da análise de western blot para marcadores exosomal e PCR para mRNAs de interesse. Microscopia eletrônica de transmissão (TEM) e immunogold rotulagem será usado para validar exosomal morfologia e integridade. O RNA total será purificada a partir destes exosomes para assegurar que podemos estudar tanto mRNA e miRNA partir da mesma amostra. Depois de validar a integridade do RNA por Bioanalyzer, vamos realizar um meio de transferência quantitativa PCR em tempo real (qPCR) para identificar o miRNA exosomal usando Taqman Matriz de Baixa Densidade (TLDA) cartas e estudos de expressão gênica de transcrições de interesse. Estes protocolos podem ser utilizados para quantificar as alterações na miRNAs exosomal in pacientes, modelos de roedores e meios de cultura de células antes e depois da intervenção farmacológica. Conteúdo exosomal variar devido à fonte de origem e as condições fisiológicas de células que secretam exossomos. Essas variações podem fornecer uma visão sobre como as células e os sistemas de lidar com o estresse ou perturbações fisiológicas. Nossos dados representativos mostram variações em miRNAs apresentam em exossomos purificados a partir do sangue mouse, sangue humano e os meios de cultura de células humanas

Após o isolamento exosomes de meios de cultura de sangue ou de células, a pureza das exosomes pode ser testado por microscopia electrónica (ME) e Western blot (Figuras 1A e 1B). Confirmámos a nossa preparação exosome partir de várias fontes com EM e western blot utilizando anticorpos múltiplos. Figura 1A mostra imagens EM confirmando que exosomes estão intactas com um diâmetro de 30 ~ -100 nm e contêm CD81 por imuno rotulagem. Marcadores exosomal comumente ut...

Watch this Scientific Journal Video about Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media at JoVE.com

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

A presença de microRNAs (miRNAs estáveis) em exossomos gerou enorme interesse como um novo modo de comunicação intercelular, pela sua utilidade potencial como biomarcadores e como uma rota para a intervenção terapêutica. Aqui demonstramos purificação exosome de mídia e cultura de sangue, seguido por PCR quantitativo para identificar os miRNAs sendo transportados.

Purificação e microRNA Profiling de Exosomes derivados do sangue e Meios de Cultura

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

purification-microrna-profiling-exosomes-derived-from-blood-culture

Research

JoVE Journal

Biology

31.7K visualizações.  Drexel University College of Medicine. A presença de microRNAs (miRNAs estáveis) em exossomos gerou enorme interesse como um novo modo de comunicação intercelular, pela sua utilidade potencial como biomarcadores e como uma rota para a intervenção terapêutica. Aqui demonstramos purificação exosome de mídia e cultura de sangue, seguido por PCR quantitativo para identificar os miRNAs sendo transportados.

Vídeo: Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

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 Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e cultura de células endoteliais pulmonares de camundongos Neonatal

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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 ...

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 ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Caracterização fenotípica e funcional das células endoteliais Formadoras de Colônias derivado do sangue do cordão umbilical humano

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Purificação de Transcrições e metabolitos de<em&gt; Drosophila</em&gt; Cabeças

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolamento de Blood-derivado do navio Multipotentes Precursores do músculo-esquelético humano

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

Perfis de expressão de microRNA iPS Human Cells, Retinal Pigment Epithelium Derivado de Ips, e Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Geração e cultura de sangue Conseqüência células endoteliais do sangue periférico humano

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 &#176;C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

Purificação de plaquetas de sangue de rato

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), ...