Este estudio fue apoyado por fondos de subvención de la Fundación Rita Allen a Seena Ajit. Los autores desean agradecer a Erika Balogh y el Dr. Soumitra Ghoshroy de la Universidad de Carolina del Sur Centro de Microscopía Electrónica para el uso de instrumentos, la asistencia científica y técnica.

Todos los experimentos con muestras de sangre de humanos y roedores fueron ejecutados en cumplimiento de todas las normas, reglamentos y las agencias reguladoras. Los sujetos humanos se inscribieron después de dar su consentimiento informado aprobado por la Drexel University College de la Junta de Revisión Institucional de la medicina y los procedimientos de los estudios realizados con animales fueron aprobados por el Cuidado de Animales institucional de Drexel y el empleo Comisión. 1. La purificación Exosome de sangre (~ 5,5 h) CONSEJOS <ul><li class="jove_content"> Nos Resuspender exosomas de un tubo de sangre (~ 10 ml de sangre humana y ~ 2 ml de sangre del ratón) en 300 l de ARN tampón de lisis del kit de aislamiento de los genes miARN mirVana. </li><li class="jove_content"> Para purificar exosomas de medios de cultivo celular, el medio se centrifugó a 500 xg durante 10 min para eliminar las células y también se filtra a través de un filtro de 0,22 micras después de la centrifugación a 12.000 x g. La purificación de exosomas de medios de cultivo celularno requiere una dilución en PBS, el lavado con PBS o el segundo paso ultracentrifugación. </li><li class="jove_content"> Si se utilizan los exosomas purificados para los estudios de proteómica, la adición de una etapa de centrifugación en gradiente de sacarosa tras la etapa de ultracentrifugación se reducirá la cantidad de agregados de proteínas y contaminantes 15. </li><li class="jove_content"> La morfología observada de exosomas puede diferir dependiendo de la fuente y los pasos de procesamiento para TEM. No se observó la morfología en forma de copa de exosomas que se ha atribuido previamente a las diferencias en el procesamiento de muestras 9,15. Nuestras imágenes de TEM, incluyendo la caracterización inmuno son similares a los anteriores informes de diferentes fuentes, incluyendo sangre, biliar y la cultura cortical exosomas derivados 9,16,17. </li></ul><ol><li> Invierta el tubo recubierto EDTA que contiene el 5x y lugar de sangre en posición vertical a temperatura ambiente durante 10 a 60 min. </li><li> Centrifugar a 2000 xg durante 15 min a 4 ° C. </li&gt; <li> Recoger la capa superior, el plasma, en tubos de 15 ml y mantener en hielo. </li><li> Diluir fluido con un volumen igual de PBS. Centrifugar 30 minutos a 2000 xg a 4 ° C. </li><li> Transferir a tubos de centrífuga, añadir 1X PBS para un volumen total de 24 ml, y se centrifuga 45 min a 12.000 xg a 4 ° C. </li><li> Traslado al ultracentrifugar tubos y centrifugar 2 horas a 110.000 xg a 4 ° C. </li><li> Resuspender el pellet en PBS y centrifugar 1 hora a 100.000 xg a 4 ° C. </li><li> Resuspender el sedimento en tampón apropiado (tampón de lisis de ARN). </li><li> Volver a suspender en tampón PBS o RIPA para microscopía electrónica y análisis de transferencia Western, respectivamente. </li></ol> 2. Purificación de ARN utilizando un kit de aislamiento de los genes miARN mirVana Modificado (~ 1 hora) CONSEJOS <ul><li class="jove_content"> Prepare el área para aislar ARN mediante el uso de equipo de protección personal (PPE), limpiar las superficies y pipetas con RNasa Zap, y el uso de puntas con filtro estériles y tubos. </li><liclass = &quot;jove_content&quot;&gt; Aunque el protocolo del proveedor da la opción de purificar sólo miRNA, siempre nos aislamos ARN total como mRNA y Mirna para que podamos estudiar tanto de la misma muestra. </li><li class="jove_content"> Adición de una etapa de tratamiento con DNasa en columna elimina los rastros de ADN que pueden estar presentes. Dependiendo de la fuente de exosomas, el ADN ha sido informado de que se ausente en exosomas purificados a partir de algunas fuentes, incluyendo ratón y líneas de células cebadas humanas 8, pero las secuencias de ADN cromosómicas han sido detectados en exosomas purificados a partir de cultivos de cardiomiocitos de los medios de comunicación 18. </li><li class="jove_content"> Hemos reducido el volumen de solución de lavado 1 a 350 l (Paso 2.4 a continuación) debido a que el proveedor proporciona sólo lo suficiente Solución de lavado 1 para uno 700 l adición. Si no se reduce el volumen no es suficiente solución de la izquierda para el uso de todo el contenido del kit. Pasos restantes se realizaron de acuerdo a las recomendaciones del fabricante. </li></ul><ol><li> Añadir 1/10 voLume de los genes miARN Aditivo homogeneizado (30 l) y se incuba en hielo durante 10 min. </li><li> Se extrae con un volumen de ácido-fenol: cloroformo igual al volumen inicial de lisado. Centrifugar y recuperar la fase superior en un tubo nuevo. </li><li> Añadir 1,25 volúmenes de 100% de etanol (375 l). Vortex. Aplicar filtro de cartucho y centrifugar 15 segundos a 10.000 x g. </li><li> Añadir la mitad de la recomendada volumen miRNA Wash Solution 1 (350 l) y centrifugar 15 segundos a 10.000 x g. </li><li> Añadir 10 l de DNasa 1 a 70 l de tampón RDD (Qiagen) para cada muestra. Añadir 80 l a la columna y se incuba 15 minutos a temperatura ambiente. </li><li> Añadir la solución de lavado 1 (350 l) y centrifugar 15 segundos a 10.000 x g. </li><li> Añadir la solución de lavado 2/3 (500 l) y centrifugar 15 seg a 10.000 x g. REPETIR. </li><li> Eluir con 40 l 95 ° C libre de nucleasa H 2 O. </li><li> Determinar la concentración de ARN y diluir los ARN a 100 ng / l. </li></ol> 3. miARN perfiles de exosomas de Blood Utilizando Taqman matriz de baja densidad (TLDA) Tarjetas (~ 6,5 h) CONSEJOS <ul><li class="jove_content"> Prepare el área para aislar ARN mediante el uso de PPE, limpiar las superficies y pipetas con RNasa Zap, y el uso de puntas con filtro estériles y tubos. </li><li class="jove_content"> Se recomienda el paso de pre-amplificación (pre-amp) de 1-350 ng RNA (350-1,000 ng muestras no requieren etapa de pre-amplificador después de la síntesis de ADNc). Es importante determinar si el pre-amplificador es necesario para la muestra, ya que debe tratar a todas las muestras en condiciones de igualdad. Etapa Pre-amp añade costos y los valores de Ct tener una de corte aceptable más bajo (32 en lugar de 40). </li><li class="jove_content"> Es útil para preparar la reacción de ADNc en tiras de tubos en lugar de una placa en función del número de tarjetas que se pueden ejecutar en un solo día. Esto minimizará la congelación y descongelación de las muestras de cDNA que no se pueden procesar en el mismo día. </li><li class="jove_content"> Los exosomas recogidos de diferentes fuentes tienen difealquilar cantidades de ARN. Este protocolo permite que un máximo de 3 l molde de ARN para la reacción de síntesis de ADNc. Dependiendo de la fuente, la cantidad de ARN exosomal que se puede aislar tal vez limitado. Mientras que un tejido típico daría suficiente RNA cDNA que se harían a partir de 100 ng de ARN en 3 l de volumen, exosomas de la sangre humana o de los medios de cultivo de células humanas a menudo contienen menos de RNA y cDNA se debe hacer a partir de material de partida menos. Exosomas sangre de ratón contienen niveles más altos de ARN y al menos 100 ng en 3 l se pueden obtener. </li></ul><ol><li> Para preparar ADNc a partir de ARN utilizando Pools Megaplex purificada transcriptasa inversa primers (Grupo A y Grupo B contienen cebadores para los miRNAs en las tarjetas de microfluidos Array A y B), descongelar los componentes de la reacción en hielo. </li><li> Label dos tubos ml RNasa libre de 1,5 para la reacción RT con Piscina A primers o cebadores Grupo B y agregar componentes a fin gráfico. Añadir al menos un volumen extra de componentes para 2 o más reacciones o hacer una mezcla maestra según lo recomendado por tque vendedor. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Componente reacción de RT </td><td> Componente Concentración </td><td> Volumen de muestra 1 (total de V / muestra = 4,5 l) </td></tr><tr><td> Agua libre de nucleasa </td><td></td><td> 0,20 l </td></tr><tr><td> RT buffer Megaplex (10 veces) </td><td> 1X </td><td> 0,80 l </td></tr><tr><td> dNTPs (100 mM) </td><td> 4,4 mM </td><td> 0,20 l </td></tr><tr><td> MgCl2 (25 mM) </td><td> 5 mM </td><td> 0,90 l </td></tr><tr><td> RNasa Inhibidor (20U/μl) </td><td> 2 U </td><td> 0,10 l </td></tr><tr><td> Megaplex RT cebadores A o B (10X) </td><td> 1X </td><td> 0,80 l </td></tr><tr><td> MultiScribe de transcriptasa inverso </td><td> 75 U </td><td> 1,50 l </td></tr></tbody></table><ol start="3"><li> Invertir tubos de 6 veces y cientorifuge brevemente. Entonces, una pipeta 4,5 l mezcla de reacción de RT en MicroAmp Tiras de 8 tubos o un plato de reacción óptica de 96 pocillos MicroAmp. </li><li> Añadir 100 ng de RNA y ajustar el volumen de 3 l de agua tratada con DEPC o añadir 3 l de ARN total, revuelva con la punta de la pipeta, y sellar los tubos o placas. Girar brevemente e incubar en hielo durante 5 min. </li><li> Cargue la reacción en la máquina PCR y ejecutarla en las condiciones siguientes (según lo recomendado por el protocolo Biosystems Pools Megaplex Aplicada): </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Etapa </td><td> Temperatura </td><td> Tiempo </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> Sostener </td><td> 85 ° C </td><td> 5 min </td></tr><tr><td> Sostener </td><td> 4 ° C </td><td> ∞ </td></tr></tbody></table><ol start="6"><li> Guarde cDNA a -20 ° C (bueno por lo menos una semana) o seguir paso pre-Amp que se puede almacenar durante una semana. </li><li> Descongele primers preamplificador para el Grupo A y el Grupo B en el hielo e invierta para mezclar. Remolino Taqman Pre-Amp Master Mix y mantener en hielo también. </li><li> Siga la tabla añadiendo al menos un volumen extra de componentes para 2 o más reacciones o hacer una mezcla maestra según lo recomendado por el proveedor. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Componente Reacción Pre-Amp </td><td> Volumen de muestra 1 </td></tr><tr><td> Agua libre de nucleasa </td><td> 7,5 l </td></tr><tr><td> TaqMan PreAmp Master Mix (2X) </td><td> 12,5 l </td></tr><tr><td> Primers preamplificador Megaplex A o B (10 veces) </td><td> 2,5 l </td></tr></tbody></table><ol start="9"><li> Pipetear 2,5 l RT product en MicroAmp tiras 8-tubo o una placa de reacción óptica de 96 pocillos MicroAmp y dispensar 22,5 l de la mezcla de reacción PreAmp en cada pocillo. </li><li> Tapar la placa e incubar en hielo 5 min y luego cargar la muestra en la máquina de PCR. Ejecutar en las siguientes condiciones. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Etapa </td><td> Temperatura </td><td> Tiempo </td></tr><tr><td> Sostener </td><td> 95 ° C </td><td> 10 min </td></tr><tr><td> Sostener </td><td> 55 ° C </td><td> 2 min </td></tr><tr><td> Sostener </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> Sostener </td><td> 99,9 ° C </td><td> 10 min </td></tr><tr><td> Sostener </td><td> 4 ° C </td><td> ∞ </td></tr></tbody></table><ol start="11"><li> Después de que se terminó la reacción preamplificador, centrifugar brevemente los tubos o la placa y a continuación, añadir 75 l 0,1 X TE pH 8,0 a cada pocillo o tubo. </li><li> Selle y mezclar y centrifugar brevemente. Almacenar el producto diluido durante una semana a -20 ° C o continuar para cargar el plato. </li><li> Combina lo siguiente en un tubo de 1,5 ml RNasa libre de hielo. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Componente </td><td> Volumen 1 de la tarjeta </td></tr><tr><td> TaqMan PCR Universal Master Mix, No AmpErase UNG (2X) </td><td> 450 l </td></tr><tr><td> Producto diluido preamplificador </td><td> 9 l </td></tr><tr><td> Agua libre de nucleasa </td><td> 441 l </td></tr></tbody></table><ol start="14"><li> Invierta el tubo para mezclar y centrifugar brevemente. Dispensar 100 l de la mezcla de reacción de PCR en cada puerto de la tarjeta de matriz de microARN Taqman. </li><li> Se centrifuga la tarjeta de 2 x 1000 g durante 1 min. Sellar la tarjeta y ejecutarlo usando la plantilla proporcionada con los iniciadores del manual de Applied Biosystems y así bajo la configuración de matrices de densidad 384 Taqman. Utilizamos Applied Biosystems 7900HT Fast System PCR en tiempo real. </li></ol> 4. Análisis de ARNm por Taqman (~ 1,5 h) <ol><li> Para preparar reacción qPCR, mezclar los componentes de la reacción en el orden en el gráfico. Todas las muestras se analizaron con sondas de cebadores para el gen de interés, así como ARNr 18S como el gen normalizador. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Taqman componente de reacción </td><td> Volumen de muestra 1 </td></tr><tr><td> Agua libre de nucleasa </td><td> 7 l (ajuste sobre la base de la muestra individual) </td></tr><tr><td> Taqman rápida Master Mix (2X) </td><td> 10 l </td></tr><tr><td> Primer sondas Taqman (20x) </td><td> 1 l </td></tr><tr><td> ADNc a partir de 100 ng de ARN </td><td> 2 y mu; L (ajustar sobre la base de la muestra individual) </td></tr></tbody></table><ol start="2"><li> Ejecutar la placa de 96 pocillos usando el rápido bloque de 96 pocillos como se recomienda por el fabricante (Biosystems 7900HT Fast sistema de PCR en tiempo real Aplicada). </li></ol>

<ol>
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Los autores tienen nada que revelar.

En este protocolo, se muestra la cuantificación de miRNAs y mRNAs de exosomas purificados por centrifugación diferencial a partir de la sangre y los medios de cultivo. Los exosomas tienen diversos componentes que dependen de su origen y están implicados en un número de funciones biológicas, incluyendo la respuesta inmune, la presentación de antígenos, la comunicación intracelular, y la transferencia de ARN y proteínas 9,11,12,19,20. Aunque el tamaño y la forma es un factor determinante de la pureza ...

MiRNAs no codificantes cortos modulan la expresión de genes mediante la unión al ARNm diana. Semilla complementariedad de secuencia de ~ 7 pares de bases permite a los genes miARN para unirse al ARNm diana que resulta en la inhibición de la traducción o en la reducción en la estabilidad del ARNm, ambos de los cuales pueden resultar en una disminución de expresión de la proteína diana 1. Las investigaciones realizadas durante la última década se ha demostrado de manera inequívoca un papel fundamental para los miRNAs en la mediación de las funciones celulares. También ha habido un considerable esfuerzo dirigido hacia la disección de los genes miARN cambios moleculares que subyacen a diversas enfermedades mediadas por 2,3. Por otra parte, la reciente identificación de miRNAs estables en los fluidos corporales 4-6 allanó el camino para su uso como nuevos biomarcadores susceptibles de diagnóstico clínico. Un medio de transporte miRNA en los líquidos corporales es a través de los exosomas, pequeñas vesículas que transportan los ARNm, proteínas, mediadores lipídicos, y miRNAs a las células receptoras a través de la sangre circulante sistémica7-14 de iones. Esto resulta en la modulación de la expresión génica en las células receptoras y representa un nuevo mecanismo de comunicación celular. Por ejemplo, las células pueden modular procesos de inmuno-reguladoras mediante la secreción y / o absorbentes de exosomas que contienen biomoléculas implicadas en la inflamación tales como la interleuquina-1β (IL1), factor de necrosis tumoral-α (TNF), factor de crecimiento transformante-β5 (TGFβ5), y los miRNAs que regulan estos genes 13. Como los genes miARN expresión aberrante es una característica común en una variedad de enfermedades humanas, estas moléculas ofrecen nuevas oportunidades para el descubrimiento y validación de nuevas dianas terapéuticas 2. MiRNAs circulantes están presentes en todos los fluidos corporales y se sabe que la composición de los exosomas es diferente en función de las celdas de origen de donde fueron liberados. Por lo tanto ofrecen una vía para estudiar el estado fisiológico de las células y cómo las células alteran la señalización inclusots en respuesta al estrés incluyendo enfermedades. El estudio de las alteraciones en la composición exosome puede dar una idea de la transducción de señales e investigar su posible utilidad como biomarcadores o vías de intervención terapéutica. Aquí vamos a demostrar la purificación de los exosomas de múltiples fuentes basadas en protocolos publicados. Estos exosomas serán utilizados para el aislamiento de ARN seguido por qPCR para identificar y medir los niveles de miRNAs presentes en los exosomas.

<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 estables están presentes en todos los fluidos corporales y algunos miRNAs circulantes están protegidos de la degradación por secuestro en pequeñas vesículas llamadas exosomas. Los exosomas pueden fusionarse con la membrana plasmática que resulta en la transferencia de ARN y proteínas a la célula diana. Sus funciones biológicas incluyen la respuesta inmune, la presentación de antígenos, y la comunicación intracelular. Entrega de miRNAs que pueden regular la expresión génica en las células receptoras a través de la sangre ha abierto nuevas vías para la intervención de destino. Además de ofrecer una estrategia para la administración de fármacos o agentes terapéuticos de ARN, contenido exosomal pueden servir como biomarcadores que pueden ayudar en el diagnóstico, la determinación de las opciones de tratamiento y el pronóstico. Aquí vamos a describir el procedimiento para el análisis cuantitativo de miRNAs y ARN mensajeros (ARNm) a partir de exosomas secretados en la sangre y de los medios de cultivo de células. Exosomas purificados se caracterizaron mediante análisis de Western blot para exosmarcadores Omal y PCR para ARNm de interés. Microscopía electrónica de transmisión (TEM) y inmunomarcaje se utilizan para validar la morfología y la integridad exosomal. Total de ARN se purificaron a partir de estos exosomas para asegurar que se puede estudiar tanto mRNA y miRNA de la misma muestra. Después de validar la integridad del ARN por Bioanalyzer, vamos a realizar una producción de PCR cuantitativa en tiempo real medio (qPCR) para identificar los genes miARN exosomal utilizando Taqman matriz de baja densidad (TLDA) cartas y estudios de expresión génica de las transcripciones de interés. Estos protocolos pueden utilizarse para cuantificar los cambios en los miRNAs exosomal en pacientes, modelos de roedores y medios de cultivo de células antes y después de la intervención farmacológica. Contenido exosomal varían debido a la fuente de origen y las condiciones fisiológicas de las células que secretan exosomas. Estas variaciones pueden dar una idea de cómo las células y los sistemas de lidiar con el estrés o perturbaciones fisiológicas. Nuestros datos representativos muestran variations en miRNAs presentes en los exosomas purificados a partir de sangre de ratón, sangre humana y los medios de cultivo de células humanas. Aquí vamos a describir el procedimiento para el análisis cuantitativo de miRNAs y ARN mensajeros (ARNm) a partir de exosomas secretados en la sangre y de los medios de cultivo de células. Exosomas purificados se caracterizaron mediante análisis de Western blot para detectar marcadores exosomal y PCR para ARNm de interés. Microscopía electrónica de transmisión (TEM) y inmunomarcaje se utilizan para validar la morfología y la integridad exosomal. Total de ARN se purificaron a partir de estos exosomas para asegurar que se puede estudiar tanto mRNA y miRNA de la misma muestra. Después de validar la integridad del ARN por Bioanalyzer, vamos a realizar una producción de PCR cuantitativa en tiempo real medio (qPCR) para identificar los genes miARN exosomal utilizando Taqman matriz de baja densidad (TLDA) cartas y estudios de expresión génica de las transcripciones de interés. Estos protocolos pueden utilizarse para cuantificar los cambios en los miRNAs exosomal in los pacientes, los modelos de roedores y medios de cultivo de células antes y después de la intervención farmacológica. Contenido exosomal varían debido a la fuente de origen y las condiciones fisiológicas de las células que secretan exosomas. Estas variaciones pueden dar una idea de cómo las células y los sistemas de lidiar con el estrés o perturbaciones fisiológicas. Nuestros datos representativos muestran variaciones en miRNAs presentes en exosomas purificados a partir de sangre de ratón, sangre humana y de los medios de cultivo de células humanas

Después de aislamiento de exosomas de la sangre o los medios de cultivo de células, la pureza de los exosomas puede ser probado por microscopía electrónica (EM) y western blot (Figuras 1A y 1B). Se confirmó nuestras preparaciones de exosoma de diversas fuentes con EM y Western blot utilizando anticuerpos múltiples. Figura 1A muestra EM imágenes que confirma que los exosomas son intacta, con un diámetro de ~ 30 -100 nm y contienen CD81 por medio del etiquetado in...

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

La presencia de microRNAs estables (miRNAs) en exosomas ha generado gran interés como un nuevo modo de comunicación intercelular, por su utilidad potencial como marcadores biológicos y como una ruta para la intervención terapéutica. Aquí se demuestra la purificación de exosomas sanguíneos y medios de cultivo seguido de PCR cuantitativa para identificar miRNAs que se transportan.

Purificación y microRNA perfiles de exosomas derivados de la sangre y Cultura Medios

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 vistas.  Drexel University College of Medicine. La presencia de microRNAs estables (miRNAs) en exosomas ha generado gran interés como un nuevo modo de comunicación intercelular, por su utilidad potencial como marcadores biológicos y como una ruta para la intervención terapéutica. Aquí se demuestra la purificación de exosomas sanguíneos y medios de cultivo seguido de PCR cuantitativa para identificar miRNAs que se transportan.

Video: 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.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

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

Investigación

Biología

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.

Aislamiento y cultivo de células endoteliales pulmonares de ratones recién nacidos

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.

Aislamiento y caracterización de ARN que contienen exosomas

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.

Aislamiento y purificación de Kinesin desde Drosophila Los embriones

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.

Caracterización fenotípica y funcional de las células endoteliales Formadoras de Colonias derivados de la sangre del cordón umbilical

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.

Purificación de las transcripciones y los metabolitos de<em&gt; Drosophila</em&gt; Cabezales

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.

Aislamiento de Vaso sanguíneo derivado Multipotentes Precursores de 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.

Perfiles de expresión de microARN de las células iPS humanas, de la retina del epitelio pigmentario Derivado de Ips y Fetal epitelio pigmentario de la retina

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.

Generación y Cultura de Sangre Consecuencia endoteliales Las células de la sangre periférica humana

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.

Purificación de plaquetas de la sangre del ratón

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