Name: isolation and profiling of microrna-containing exosomes from human bile
Uploaded: 2016-06-13T14:00:00.000+00:00
Duration: 6 min 59 s
Description: Watch this Scientific Journal Video about Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile at JoVE.com

This study was supported by a K08 Award (DK090154-01) from the National Institutes of Health (NIH; to F.M.S.).

La obtención de la bilis de pacientes por la colangiopancreatografía retrógrada endoscópica (CPRE) requiere la aprobación de un protocolo de estudio los sujetos humanos por la Junta de Revisión Institucional. Todo el trabajo que aquí se presenta ha sido aprobado por la Junta de Revisión Institucional de la Universidad Johns Hopkins. 1. Aislamiento de exosomas de la bilis <ol><li> Realizar la colangiopancreatografía retrógrada endoscópica (CPRE). Colocar al paciente en posición supina y intubar. <ol><li> Pasar un duodenoscopio a través de la boca del paciente y la inserta en la segunda parte del duodeno e identificar la papila mayor. </li><li> Selectivamente canular el conducto biliar común mediante la inserción de un triple lumen esfinterotomo pre-cargado con una guía hidrofílica 0,035 pulgadas (0,9 mm). </li><li> Avanzar la guía bajo guía fluoroscópica con el conducto hepático izquierdo ya que en ocasiones puede ser difícil diferenciar el conducto cístico desde el conducto hepático derecho, a continuación, hacer avanzar elesfinterótomo 5 cm en el conducto biliar para adquirir una posición estable. </li><li> Retire el alambre guía, acople una jeringa de 10 ml al puerto de alambre guía a través del cierre Luer y aspirar la bilis utilizando 10 ml de presión negativa. </li><li> Retire la jeringa que contiene la bilis para proceder con la CPRE volviendo a insertar el alambre de guía y el agente de contraste a través de la inyección de la abertura de inyección para opacificar el árbol biliar. </li><li> La transferencia de la bilis recogido en un tubo de 15 ml en hielo y mantener en hielo hasta que esté listo para proceder con la etapa de centrifugación. Precaución: Use guantes para protegerse y proteger a las muestras! El tratamiento de la bilis como una fuente potencial de infección, ya que puede contener partículas virales de hepatitis A! </li><li> Mantenga la muestra (s) en hielo. Cualquiera de proceder con el paso 1.2 o centrifugar a 500 xg a 4 ° C durante 10 min y luego se congelan a -80 ° C hasta su uso posterior. Nota: Las muestras almacenadas de esta manera durante varios años de forma rutinaria se han utilizado con buenos resultados 19. </li></ol&gt; </li><li> Transferencia de 400 l de bilis en un tubo de microcentrífuga y recoger las células y los restos celulares por centrifugación a 300 xg durante 10 min a 4 ° C. </li><li> Eliminar el sobrenadante con la pipeta, transferir a un tubo de microcentrífuga fresco y centrifugar la muestra (s) a 16.500 xg durante otros 20 min a 4 ° C para borrar el sobrenadante de desechos adicionales. Desechar los tubos en los residuos de riesgo biológico. Como alternativa, girar las muestras en una ultracentrífuga lugar. </li><li> Recoger el sobrenadante y filtrar en una cabina de bioseguridad con una jeringa a través de un filtro de membrana de 0,2 micras de polietersulfona (PES) que tiene buenas tasas de flujo y baja en proteínas de unión para eliminar las partículas más grandes que 200 nm fue. </li><li> Pipetear el sobrenadante se filtró para ultracentrífuga tubos y añadir PBS de modo que los tubos son más de 2/3. Si los tubos contienen menos líquidos que eso, los tubos se derrumbará durante la centrifugación! Los tubos pueden ser lavados, en autoclave y volver a utilizar varias veces. </li><li> Pelly los exosomas mediante ultracentrifugación a 120.000 xg durante 70 min a 4 ° C. Después de la centrifugación mirada en la parte inferior del tubo de ultracentrífuga para ver a veces pequeñas bolitas amarillas; estos son los exosomas. </li><li> Eliminar el sobrenadante por decantación con cuidado y desinfectar los residuos mediante la adición de cloro para una concentración final de 10% v / v y se deja reposar durante 20 minutos. </li><li> Para los experimentos de aguas abajo, o bien procesar la pastilla (s) en un pequeño volumen de la solución apropiada (es decir, ensayo de radioinmunoprecipitación (RIPA) tampón para el aislamiento de proteínas, tampón de lisis de ARN para el aislamiento MIR) o resuspender en 50 a 150 l de fosfato tamponada de solución salina (PBS) que puede ser almacenado, ya sea a -80 ° C para su uso futuro (reacción en cadena de polimerasa cuantitativa en tiempo real (qRT-PCR) o la extracción de proteínas etc.) o se utiliza para la caracterización de la preparación exosome por TEM o análisis óptico de nanopartículas . </li></ol> 2. Identificación por exosomeMicroscopía Electrónica de Transmisión (TEM) o cryoEM <ol><li> Volver a suspender las vesículas extracelulares en 100 l de PBS con la pipeta. Lo ideal es utilizar exosomas que no han sido almacenadas a -20 ° C o -80 ° C, especialmente cuando se realiza el aislamiento por primera vez. </li><li> Adsorber una alícuota de 20 l (aproximadamente 2 g) a 400 rejillas de cobre Parlodion recubiertas de carbono de malla durante 2 minutos y dejar secar a temperatura ambiente. </li><li> Fijar el exosoma en 1% (v / v) de glutaraldehído (EM-grado) durante 5 min a RT mediante la colocación de gotas cuidadosamente sobre la preparación seca. </li><li> Lavar las rejillas dos veces con agua durante 5 min y luego contrastar EVs mancha con ácido fosfotúngstico al 1% (PTA) durante 30 s. </li><li> Adquirir imágenes con un microscopio electrónico con un voltaje de aceleración de 80 kilovoltios a partir de las ampliaciones de 20.000X y se eleva a 100,000X al determinar el tamaño de las partículas. Nota: Otras preparaciones como capas en 200 rejillas de malla de carbono recubiertos con Formvar de cobre o níquel y li contraste tinciónke 1% o 2% de acetato de uranilo durante 10-15 min también son posibles. </li><li> Para cryoEM, mezclar una parte alícuota de 20 l (aproximadamente 2 g) con 60 l de proteína G de oro 10 nm (relación 1: 3). Esto ayudará a determinar el diámetro de exosomas. </li><li> La transferencia de las muestras sobre rejillas de carbón perforado descargada resplandor-C-plana y eliminar el exceso de líquido mediante secado. Congelar las rejillas sumergiéndolos inmediatamente en etano líquido. </li><li> Montar las rejillas en un cryoholder en nitrógeno líquido. </li><li> Adquirir las imágenes en 200 kV con magnificaciones de 20.000X y 100,000X. </li></ol> 3. Identificación por exosome multiparámetros de nanopartículas análisis óptico <ol><li> Volver a suspender los exosomas aislados en 100 l de PBS con la pipeta. </li><li> Diluir exosomas en 600 l de PBS en varias proporciones diferentes (como 1:50, 1: 100, 1: 300, 1: 600 y 1: 1200). </li><li> Visualizar los exosomas por dispersión de luz láser con el instrumento y el software adecuado en tiempo real de acuerdo con la fabriel protocolo del RER 20. </li><li> Aumentar el nivel de la cámara en el modo de captura hasta que las partículas se hacen visibles, a continuación, ajustar el enfoque para hacer que las partículas se vean sin problemas. </li><li> Mientras que en el modo estándar, disminuir la cámara al nivel más bajo que las partículas todavía pueden verse, si es necesario ajustar obturador de la cámara y la ganancia para lograr esto. </li><li> una inspección visual para asegurarse de 20-100 partículas son visibles en la pantalla, si no eliminar la unidad y ajustar la dilución en PBS. </li><li> A continuación, ajuste la duración de captura de entre 30-60 seg. Después de que se capturó el vídeo, ajustar los parámetros de proceso hasta que todas las partículas se clasifican en la pantalla y procesar el archivo de vídeo. </li></ol> 4. Caracterización de exosomas por Western Blot <ol><li> Lyse gránulos de exosoma de 2-5 ml de bilis en 100 l de ensayo de radioinmunoprecipitación enfriado con hielo (RIPA) tampón con un inhibidor de la proteasa pipeteando arriba y abajo a fondo y mezclar lisados ​​vigorosamente durante 30 secen un vórtice para solubilizar las proteínas de manera eficaz. </li><li> Mientras que la mayoría de las veces no es necesario, utilizar pulsos de sonicación de las muestras en hielo con 2 impulsos de 10 segundos cada uno para asegurar la lisis completa. </li><li> Se precipitan material insoluble por centrifugación a 15.000 xg a 4 ° C durante 5 min y transferir el sobrenadante a un tubo limpio. </li><li> Determinar la concentración de proteína con un método de elección (es decir, el ácido bicinconínico (BCA), Coomassie, Lowry modificado, 660 nm de ensayo de proteínas, etc.) según el protocolo del fabricante. El método no es tan importante como el tiempo que se utiliza el mismo método consistente para lograr los resultados que se pueden comparar a través de experimentos. </li><li> Cargar 50 g de proteína por muestra en tampón de Laemmli después de calentar la muestra a 95 ° C durante 5 min en un pozo de un gel de poliacrilamida para electroforesis (PAGE). </li><li> La electroforesis de las muestras en los geles PAGE durante 1,5 horas y transferir las proteínas separadas a una membrana de nylon. </li><li> segundobloquear la membrana (s) con 5% de leche sin grasa en solución salina tamponada con Tris con 0,1% de Tween 20 (TBS-T) durante 1 h, después se incuba con anticuerpos de exosoma específica como anti-CD63 y anti-TSG-101 a una dilución de 1: 500, preferiblemente O / N a 4 ° C. </li><li> Lavar las membranas con TBS-T 3 x 10 min, a continuación se incuba con un apropiado, búsqueda de anticuerpo secundario conjugado con HRP en 5% de leche sin grasa en TBS-T durante 1-2 horas a temperatura ambiente. </li><li> Lavar la membrana (s) 3 x 5 min con TBS-T y detectar las proteínas específicas con reactivos de quimioluminiscencia de acuerdo con el protocolo del fabricante. </li><li> Image las membranas con película o un sistema de imagen digital de acuerdo con el protocolo del fabricante. </li></ol> 5. El microARN aislamiento de los exosomas aislados Nota: El aislamiento de los genes miARN de exosomas biliares se lleva a cabo utilizando un aislamiento de los genes miARN modificado basado en un kit disponible en el mercado, pero cualquier otro método de aislamiento de ARN puede utilizarse o adaptarse. <ol><li> Añadir 300 y# 181; l tampón de lisis / edificio al sedimento de exosomas (aislado de la bilis 400 l), vórtice, pipeta o pasar a través de una aguja / jeringa para lisar completamente los exosomas para obtener un lisado homogénea. </li><li> Añadir 1/10 (30 l) volumen de miARN homogeneizado aditivo, y mezclar bien por agitación o invirtiendo el tubo varias veces. </li><li> Incubar la mezcla durante 10 min en hielo. </li><li> Añadir un volumen de ácido-fenol: cloroformo igual al homogeneizado inicial (300 l). </li><li> Añadir 5 l de 5 fmol / l cel-MIR 39 y agitar durante 30-60 segundos. </li><li> Centrifugar durante 5 min a 10.000 xg a la temperatura ambiente para separar la fase acuosa (superior) y las fases orgánicas (inferiores). </li><li> aspirar con cuidado la fase superior con la pipeta y transferir a un tubo nuevo al tiempo que observa el volumen transferido. Descartar la fase orgánica en el contenedor de residuos orgánico apropiado. </li><li> Para enriquecer a los pequeños ARN añaden 1/3 volumen de etanol al 100% a la fase acuosa recuperada y mezclar mediante agitación o invirtiendo muchísimo de la maser varias veces. </li><li> Pipetear la mezcla de lisado / etanol en el cartucho de filtro, hasta 700 l a la vez. </li><li> Centrifugar los cartuchos de 15 segundos a un máximo de 10.000 xg o aplicar vacío para pasar la mezcla a través del filtro. </li><li> Recoger el filtrado y si la mezcla de lisado / etanol es superior a 700 l, transferir el flujo a través de un tubo nuevo y repita el procedimiento para recoger todos los filtrados. Estos filtro contiene fracciones de ARN desprovistos de ARN pequeños y se puede utilizar para recuperar estos ARN de mayor tamaño. </li><li> Añadir 2/3 volumen de RT 100% de etanol al filtrado total y mezclar bien con un vórtex. </li><li> Pipetear la mezcla de filtrado / etanol en un segundo cartucho de filtro. Al igual que antes de que el volumen máximo que se puede aplicar a la vez es de 700 microlitros. </li><li> Centrifugar durante 15 segundos a 10.000 xg o aplicar vacío como se describe en el paso 5.10. </li><li> Desechar el flujo continuo, y repetir hasta que todos mezcla de filtrado / etanol ha sido filtrada. </li><li> aplicar 70081; l miARN solución de lavado 1 al cartucho de filtro, se centrifuga durante 5-10 segundos o aplicar vacío y deseche el flujo continuo. </li><li> Lavar el filtro con 500 l de solución de lavado 2/3 como se hizo en el paso 5.16. </li><li> Repetir el lavado por segunda vez con 500 l de solución de lavado como en el paso 2/3 5.16, desechar el flujo a través y colocar el filtro en el tubo de recogida. </li><li> Girar el cartucho de filtro durante 1 min a 10.000 xg para eliminar todo el fluido residual desde el filtro. </li><li> Transferir el cartucho de filtro en un tubo de recogida fresco y aplicar 100 l de agua libre (95 ° C) nucleasa caliente para el centro del filtro. </li><li> Girar los montajes de 20-30 segundos a la máxima velocidad para recuperar el ARN. </li><li> Recoger el eluido y utilizar inmediatamente o almacenar a -80 ° C. </li></ol>

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None of the authors have competing financial interests.

Para utilizar de forma fiable exosomas aisladas de la bilis, es importante emplear los métodos de aislamiento de alta calidad consistentes para obtener muestras de alta calidad a cambio. La metodología definida en este trabajo es una manera bien establecida para aislar los exosomas y miARN de la bilis humana. En él se destacan varios pasos cruciales en la caracterización de los exosomas aisladas que como mínimo deberían formar parte de microscopía electrónica de análisis óptico o de nanopartículas y transfere...

Al igual que otros fluidos biológicos, es decir, la leche materna, plasma u orina, bilis contiene lípidos exosomas, vesículas ricas 1-4. Los exosomas pueden inducir o alterar las funciones biológicas en células receptoras 5, 6, una forma de comunicación célula-célula que podría ser parte de su función normal 4, 7-9. Los exosomas pueden contener especies miARN que pueden proporcionar una valiosa fuente de biomarcadores para el diagnóstico 10. perfiles de miRNAs han ganado considerable atención en los últimos años como marcadores de diagnóstico y pronóstico para una variedad de enfermedades 11-14. sí miARN puede encontrarse en fluidos biológicos, posiblemente liberada por las células muertas y la cantidad de células que se desprenden pueden ser influenciados en gran medida por una enfermedad subyacente. El perfil de miARN de exosomas no refleja necesariamente el perfil de miRNA de las células originarias 6, 10, 15-17, sin embargo, puede llevar a exosomas firmas de miARN que podrían ser característico de la CE de los padreste gusta una célula tumoral. Exosomas derivado de un tumor se han identificado en el plasma de pacientes con adenocarcinoma de pulmón, cáncer de próstata y otros tumores 8, 16, 18. El objetivo de este método es el aislamiento fiable y robusto de exosomas de muestras biliares humanos, en gran parte independientes de sus procedimientos de manipulación anteriores. Fue desarrollado para utilizar bilis como una fuente fiable de miARN para el diagnóstico potencial de las enfermedades de las vías biliares tales como colangiocarcinoma 19. Consta de varias etapas de centrifugación con el aumento de las velocidades de aislar exosomas y podría ser aplicable a otros fluidos biológicos.

<table><tbody><tr><td>0.2 &amp;micro;m Polyethersulfone (PES) membrane filter</td><td>Corning</td><td>431229</td><td></td></tr><tr><td>thinwall polyallomer ultracentrifuge tubes&amp;nbsp;</td><td>Beckman Coulter</td><td>331374</td><td></td></tr><tr><td>SW40Ti rotor</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>LM10-HS&amp;nbsp;</td><td>Nanosight</td><td></td><td></td></tr><tr><td>Nanoparticle Tracking Analysis (NTA) software&amp;nbsp;</td><td>Nanosight</td><td></td><td></td></tr><tr><td>mirVANA</td><td>Life Technologies</td><td>AM1560</td><td></td></tr><tr><td>Complete Protease Inhibitor</td><td>Roche distributed by Sigma-Aldrich</td><td>4693116001</td><td></td></tr><tr><td>Immobilon PSQ</td><td>Millipore, Bedford MA</td><td>ISEQ00010</td><td></td></tr><tr><td>Anti-CD63 antibody</td><td>Abcam</td><td>ab59479</td><td></td></tr><tr><td>Anti-Tsg101</td><td>Abcam</td><td>ab30871</td><td></td></tr></tbody></table>

isolation and profiling of microrna-containing exosomes from human bile

exosome la investigación en los últimos tres años ha ampliado considerablemente el alcance hacia la identificación y caracterización de biomarcadores y sus usos terapéuticos. Los exosomas han sido recientemente demostrado que contienen microRNAs (MIR). MIRS mismos han surgido como biomarcadores valiosos para fines de diagnóstico. Como la recogida de muestras en clínicas y hospitales es bastante variable, miARN aislamiento de la bilis toda varía sustancialmente. Para lograr perfiles de miARN robustos, precisos y reproducibles de muestras de bilis recogidos de una manera sencilla requerido el desarrollo de un protocolo de alta calidad para aislar y caracterizar los exosomas de la bilis. El método requiere varias centrifugaciones y una etapa de filtración con una etapa de ultracentrifugación final para sedimentar las exosomas aisladas. La microscopía electrónica, transferencias Western, citometría de flujo y análisis óptico de nanopartículas de múltiples parámetros, si están disponibles, son pasos cruciales para validar la caracterización de la calidad del correoxosomes. Para el aislamiento de los genes miARN de estos exosomas, Rematar el lisado con una, miRNA sintético no específica de una especie como Caenorhabditis elegans, es decir, Cel-miR-39, es importante para la normalización de la eficacia de la extracción de ARN. El aislamiento de exosomas del líquido biliar siguiendo este método permite que el éxito miARN perfiles de las muestras de bilis acumulada durante varios años a -80 ° C.

Desde exosomas son demasiado pequeños para ser detectados por microscopía regular o citometría de flujo, microscopía electrónica o análisis óptico de nanopartículas tiene que ser realizada. El análisis óptico de nanopartículas tiene la ventaja sobre la microscopía electrónica que también es cuantitativa y proporciona distribución de tamaño y la concentración. El instrumento presenta un haz de láser finamente enfocada a través de un prisma de cristal en la muestra. El m...

Watch this Scientific Journal Video about Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile at JoVE.com

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

El aislamiento y la generación de perfiles de exosomas de la bilis humana que contiene microRNA

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

isolation-profiling-microrna-containing-exosomes-from-human

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.2K vistas.  Johns Hopkins University School of Medicine. Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Video: Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

El aislamiento de las lipoproteínas de alta densidad para no codificante ARN pequeño Cuantificación

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Investigación

Bioquímica

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Digestión de proteínas, ultrafiltración y cromatografía por exclusión de tamaño para optimizar el aislamiento de exosomas de suero y Plasma de sangre humana

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

Preparación de exosomas siRNA entrega a las células cancerosas

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

Investigación sobre el cáncer

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Aislamiento, secuenciación y análisis de microRNA extracelular de las células madre mesenquimales humanas

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Genética

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Aislamiento de exosomas del plasma de VIH-1 en individuos positivos

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Inmunología e Infección

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 ...

Biología

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Aislamiento de las vesículas extracelulares del líquido de lavado broncoalveolar murino utilizando una técnica de centrifugación ultrafiltración

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

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

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

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  ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

El aislamiento de los pequeños RNAs no codificantes de suero humano

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

Método rápido y simplificado para el aislamiento alto rendimiento de procesamiento de miRNA desde muy puras lipoproteína de alta densidad

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...