Damos las gracias a Sacha Reichman y Dominique Santiard-Baron por su ayuda en la edición del protocolo de purificación de RNA.

1. Diario: Procedimiento para recuperar las muestras del Bloque Quirúrgico En el laboratorio <ol><li> Obtenga un contrato de importación de la empresa de transporte expreso. </li><li> Llenar 10 (o 25) Formas de envío a la dirección postal del laboratorio que indica la persona de contacto en el laboratorio (número de teléfono y dirección de correo electrónico). </li><li> Preparar 10 (o 25) formas muestras numeradas del 1 al 10 (o 25). Estas formas incluyen espacios dedicados a informar sobre: ​​a) la identificación anónima del paciente, b) la fecha de la cirugía y c) cualquier observación complementaria que el cirujano le gustaría añadir. Preparar 10 (o 25) sobres acolchados (150 x 210 mm). </li><li> Elaborado a partir de RNasa libre de reactivos 25 (o 65) ml de guanidina 6 M de cloruro de dietilo (DEPC) tratados con H 2 O (GHCL). </li><li> Llenar 10 o 25 números tubos de fondo 5 ml estériles de polietileno redondas con 2,4 ml de solución GHCL. </li><li> Se utiliza gas argón para llenar la parte superior de los tubos, de prensa tightly en la tapa para evitar la oxidación de la solución GHCL lo largo de varios años de almacenamiento en el armario quirúrgica. </li><li> Introducir todos los tubos de 5 ml en un ml de polipropileno tubo de fondo cónico de 50 estéril con tapón de rosca. Use un pedazo de pañuelo de papel limpio para mantener el tubo 5 ml en el tubo de 50 ml, cerrar el tubo. </li><li> Coloque los tubos de 50 ml en un estante de poliestireno y el bastidor en una caja de cartón con los sobres acolchados, las formas, las formas de envío de muestras. </li><li> Pegue las instrucciones (véase: 1.12 a 1.19) en el interior de la cubierta de la caja de cartón para facilitar su lectura. </li><li> Envíe la caja de cartón por correo a la persona de contacto en el hospital. </li></ol> En el hospital <ol start="11"><li> Trae la caja de cartón de la caja quirúrgica a la sala quirúrgica. Nota de precaución: si el procedimiento implica un paciente inmunodeprimidos, el cartón no debe ser llevado a la sala de cirugía. </li><li> Durante la cirugía, coloque el Specim retinaes numerada en 5 ml de polipropileno estéril lleno con una solución de GHCL. Cerrar bien el tubo. </li><li> Volver brevemente el tubo. </li><li> Colocar el tubo en el eje de balancín tubo de la sangre durante 10 min. </li><li> Rellene el formulario numerado ejemplo que lo acompaña. </li><li> Informe el número de identificación en el correspondiente tubo de 50 ml numerados. </li><li> Introducir el tubo de 5 ml con la pieza quirúrgica en el tubo de 50 ml, reemplazar el tejido de papel y enrosque el tubo. </li><li> Introducir el tubo y el formulario de muestra lleno en ella en uno de los sobres acolchados acompañan. Cerrarla. </li><li> Llame a la compañía de transporte express para recoger. </li></ol> 2. La purificación de ARN En el laboratorio <ol><li> Homogeneizar la muestra en su tubo de polipropileno de 5 ml con solución GHCL con un homogenizador durante 1 min mientras se mueve el tubo hacia arriba y abajo. </li><li> Añadir 270 l de acetato de potasio 2 M de pH 5,0. Agitar vigorosamente durante 10 min colocando el tubo en un agitador en suposición horizontal (420 min -1). </li><li> Centrifugar 10 minutos a 5000 rpm (6500 xg) a 20 ° C. </li><li> Aspirar suavemente la fracción soluble sin perturbar el sedimento. </li><li> Transferir a un tubo estéril de 14 ml. </li><li> Añadir 5,3 ml de Tris 100 mM, pH 8,0, N-lauroilsarcosina al 1%. </li><li> Añadir 3,2 g de cloruro de cesio (CsCl) y mezclar el tubo por agitación. </li><li> Añadir 1,8 ml de ácido CsCl / etilendiaminotetraacético (EDTA) en un tubo de centrífuga de 11 ml estéril polialómero. </li><li> Con una pipeta Pasteur estéril ml 10, transferir la solución de ARN en 1,8 ml de CsCl / EDTA por deslizamiento lentamente en el borde del tubo para evitar perturbar el cojín densidad. </li><li> Colocar los tubos (un segundo tubo que contiene los tampones sin retina si es necesario) en el rotor. </li><li> Centrifugar 24 horas a 32.000 rpm (225.000 xg) a 20 ° C. </li><li> Quite la parte superior de la solución con una pipeta Pasteur estéril y descartarlo. </li><li> Eliminar gradualmente mientras se comprueba elmomento en que el ADN (viscoso) se aspira con una segunda pipeta Pasteur estéril y descartarla. </li><li> Retirar la solución restante teniendo cuidado de no liberar el precipitado de ARN con una tercera pipeta Pasteur estéril. </li><li> Sección de la parte inferior del tubo con una llama-bisturí esterilizado, a continuación, poner la parte restante del tubo boca abajo sobre una mirada estéril. </li><li> Invierta el tubo y enjuagar delicadamente con 160 l de GHCL. </li><li> Deje que el pellet seco durante 10 min. </li><li> Resuspender el precipitado en 150 l de (Tris 10 mM, pH 7,5 a 1 mM de EDTA - 0.1% de SDS). </li><li> Transferir la solución a un tubo de microcentrífuga de 2 ml estéril, a continuación, cosechar el sedimento residual con 30 l de (Tris 10 mM, pH 7,5 a 1 mM de EDTA - 0.1% de SDS). </li><li> Añadir 150 l de (Tris 10 mM, pH 7,5 a 1 mM de EDTA). </li><li> Añadir 30 l de 3 M de acetato sódico pH 5,0, el tubo de vórtice. </li><li> Añadir 900 l de etanol al 100% (-20 ° C), el tubo de vórtice. </li><li> Coloque el tubo de 30 min en hielo que se derrite. </li><li> Centrifuge el tubo 30 min a 15.000 rpm a 4 ° C. </li><li> Aspirar delicadamente la fracción soluble, y lo descarta. </li><li> Añadir 500 l de etanol al 70% (RT), el tubo de vórtice. </li><li> Centrifugar el tubo 20 min a 15.000 rpm (18.000 xg) a 4 ° C. </li><li> Repetir la etapa de lavado (etanol al 70%). </li><li> Centrifugar brevemente y eliminar el resto de etanol con una pipeta P200. </li><li> Deje que el aire seco pellet durante 10 min. </li><li> Resuspender el precipitado en 50 l tratada con DEPC H 2 O. </li><li> Mezclar vigorosamente en vortex. </li><li> Incubar 15 min a 45 ° C en un baño de agua. </li></ol> 3. Análisis de ARN mediante electroforesis en gel <ol><li> Vierta un gel de agarosa dentro de una campana química. </li><li> En un tubo de microcentrífuga de 1,5 ml estéril, añadir 2 l de ARN a analizar y 6,4 l de tampón de preparación de muestras. </li><li> En un segundo tubo de 1,5 ml, añadir 3 l de estándares de ARN y 9,6 l de tampón de preparación de muestras. </li><li> Calentar los tubos 15 min a 65 ° C, Ponlas en hielo. </li><li> Añadir 1 l de bromuro de etidio (EB) tampón de carga sin colorante en el tubo de muestra de ARN y 1 l EB tampón de carga con colorante en el tubo de estándares de ARN. </li><li> Ejecutar el gel en 80 a 100 V dentro de una campana química en funcionamiento de amortiguación, hasta que uno de los colorantes (azul de bromofenol) alcanza 2/3 de la parte inferior del gel. </li><li> Enjuague el gel dos veces 15 minutos con 250 ml de DEPC tratada con 2x SSC. </li><li> Tome una imagen digitalizada bajo iluminación UV. </li><li> Calcular la relación entre la banda superior (23S rRNA) y la banda inferior (16S rRNA). </li></ol> 4. La purificación final de la ARN <ol><li> Ajustar el volumen de la muestra de ARN de 100 l con tratada con DEPC H2O (si es necesario). </li><li> Añadir 100 l de ácido fenol (1 ml de fenol saturado en tratada con DEPC H 2 O + 130 l de 50 mM de acetato de sodio, pH 5.2) y agitar vigorosamente. </li><li> Centrifugar el 10 min a 13.000 rpm (15.000 xg) a temperatura ambiente. </li><li> Recover con cuidado y transferir la fase acuosa (fase superior) en una novela un tubo de microcentrífuga de 1,5 ml estéril. </li><li> Añadir 10 l de acetato de sodio 3 M, pH 5,2 y 250 l de etanol al 100% (-20 ° C). </li><li> Incubar el tubo 30 min en hielo de fusión. </li><li> Centrifugar el tubo 30 min a 14.000 rpm (18.000 xg) a 4 ° C. </li><li> Aspirar cuidadosamente la fracción soluble, y lo descarta. </li><li> Añadir 500 l de etanol al 70% (temperatura ambiente), y el tubo de vórtice. </li><li> Centrifugar el tubo 20 min a 14.000 rpm (18.000 xg) a 4 ° C. </li><li> Repetir la etapa de lavado (etanol al 70%). </li><li> Centrifugar brevemente y eliminar el resto de etanol con una pipeta P200. </li><li> Deje secar al aire el precipitado durante 10 minutos. </li><li> Resuspender el precipitado en 50 l de DEPC tratada con H 2 O. </li><li> Se incuba entonces tubo de 15 min a 45 ° C. </li><li> Medir la absorbancia (Abs) a 260 nm y 280 nm en 2 l de la muestra de ARN. Se calcula la relación Abs260/Abs280. </li> &lt;li&gt; Guarde la muestra de ARN a -80 ° C (estable durante varios años). </li></ol> 5. Soluciones y Procedimientos de Limpieza <ol><li> Acetato de potasio 2 M, pH 5,0: Disolver 19,6 g de acetato de potasio en 70 ml de DEPC tratada con H 2 O. Limpiar la sonda medidor de pH por inmersión en con NaOH 1 M durante 15 min, seguido de 5 lavados con tratada con DEPC H 2 O. Ajustar el pH a 5,0 con ácido acético a continuación, ajustar el volumen a 100 ml con tratada con DEPC H 2 O. </li><li> Tris-HCl 100 mM, pH 8,0, N-lauroilsarcosina al 1%: Disolver 2 g de N-lauroilsarcosina (bajo una campana) para 40 ml de 0,5 M de Tris-base de pH 8,0. Ajustar el volumen a 200 ml con tratada con DEPC H2O) de CsCl / EDTA: 96 g de CsCl en 50 ml de 10 mM de EDTA pH 7,5 preparado con tratada con DEPC H 2 O. Esterilizar en autoclave y ajustar el volumen a 100 ml con tratada con DEPC H 2 O. Compruebe pH &lt;7,5. </li><li> Gel de agarosa al 1%: Poner 1 g de agarosa en 62 ml DEPC tratada con H 2 O. Hervir en un horno de microondas. Añadir 18 ml de 12,3 Mformaldehído, 20 ml de MOPS 5x. Vierta dentro de una campana química. </li><li> Sample Buffer prep: Para ser extemporánea preparado. Mezclar dentro de una campana química 8 l de MOPS 5x, 16 l de 12,3 M de formaldehído y 40 l de formamida. </li><li> Tampón de carga EB (sin colorantes): Para ser extemporánea preparado. Añadir 4 l 10 mg / ml de bromuro de etidio en 20 l de 80% de glicerol preparado con tratada con DEPC H 2 O. </li><li> Tampón de carga EB (con tinte): Para ser extemporánea preparado. Añadir 2 l 10 mg / ml de bromuro de etidio en 10 l de 80% de glicerol que contiene 0,25% Xylen cianol, 0,25% de azul de bromofenol con tratada con DEPC H 2 O. </li><li> El tampón: A 60 ml de MOPS 5x, añadir 54 ml de formaldehído 12,3 M y 186 ml de DEPC tratada con H 2 O. </li><li> Tratada con DEPC H 2 O: agua destilada Incubar con pirocarbonato de dietilo 0,1% v / v durante 2 horas a 37 ° C con agitación. Esterilizar en autoclave. </li><li> Limpieza del homogeneizador: Añadir 25 ml de solución de limpieza (RBS) a 1 l de DEPC-tratado H 2 O, y sumergir el eje 1 min con agitación. Incubar 2 horas a 60 ° C. Enjuague bien con DEPC tratada con H 2 O Envolver el instrumento en aluminio y poner en el cuadro de instrumentos. Envuelva la caja. Esterilizar en autoclave. </li><li> Limpieza del dispositivo de electroforesis: Lavar el aparato, la bandeja y el peine 15-pozos. Incubar 15 minutos en peróxido de hidrógeno al 3%. Enjuagar una vez con etanol al 100%. Deje secar al aire. </li><li> Limpieza de los platos de cristal: Limpie los platos de cristal RBS 2% durante toda la noche, y luego enjuague con agua destilada antes de la esterilización a 200 ° C. </li></ol>

<ol>
	<li>Mitry, D., Charteris, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+rhegmatogenous+retinal+detachment:+geographical+variation+and+clinical+associations.">The epidemiology of rhegmatogenous retinal detachment: geographical variation and clinical associations.</a> The British Journal of Ophthalmology. 94 (6), 678 (2010).</li><li>Green, R. E., Krause, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+draft+sequence+of+the+Neandertal+genome.">A draft sequence of the Neandertal genome.</a> Science. 328 (5979), 710 (2010).</li><li>Glisin, V., Crkvenjakov, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonucleic+acid+isolated+by+cesium+chloride+centrifugation.">Ribonucleic acid isolated by cesium chloride centrifugation.</a> Biochemistry. 13 (12), 2633 (1974).</li><li>Van Gelder, R. N., von Zastrow, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 87 (5), 1663 (1990).</li><li>Cavalieri, L. F., Yamaura, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=coli+tRNAs+as+inhibitors+of+viral+reverse+transcription+in+vitro.">coli tRNAs as inhibitors of viral reverse transcription in vitro.</a> Nucleic Acids Research. 2 (12), 2315 (1975).</li><li>Sawadogo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+inhibition+of+yeast+RNA+polymerases+A+and+B+by+tRNA+and+alpha-amanitin.">On the inhibition of yeast RNA polymerases A and B by tRNA and alpha-amanitin.</a> Biochemical and biophysical research communications. 98 (1), 261 (1981).</li><li>Delyfer, M. N., Raffelsberger, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomic+analysis+of+human+retinal+detachment+reveals+both+inflammatory+response+and+photoreceptor+death.">Transcriptomic analysis of human retinal detachment reveals both inflammatory response and photoreceptor death.</a> PloS ONE. 6 (12), e28791 (2011).</li><li>Kalathur, R. K., Gagniere, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RETINOBASE:+a+web+database,+data+mining+and+analysis+platform+for+gene+expression+data+on+retina.">RETINOBASE: a web database, data mining and analysis platform for gene expression data on retina.</a> BMC Genomics. 9, 208 (2008).</li><li>Nakazawa, T., Hisatomi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+chemoattractant+protein+1+mediates+retinal+detachment-induced+photoreceptor+apoptosis.">Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (7), 2425 (2007).</li><li>Leveillard, T., Sahel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rod-derived+cone+viability+factor+for+treating+blinding+diseases:+from+clinic+to+redox+signaling.">Rod-derived cone viability factor for treating blinding diseases: from clinic to redox signaling.</a> Science Translational Medicine. 2 (26), 26ps16 (2010).</li><li>Chalmel, F., Leveillard, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rod-derived+Cone+Viability+Factor-2+is+a+novel+bifunctional-thioredoxin-like+protein+with+therapeutic+potential.">Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential.</a> BMC Molecular Biology. 8, 74 (2007).</li></ol>

Los autores no tienen nada que revelar.

El desarrollo de un procedimiento para la recuperación de los tejidos del bloque quirúrgico ha sido esencial para el análisis del transcriptoma de desprendimiento de retina. Uno debe darse cuenta de que este tipo de cirugía se practica en situaciones de emergencia y que el operativo oftalmólogos tienen poco tiempo para participar en un programa de investigación biológica cuando operan. Este retinectomía también se lleva a cabo estocásticamente en cada servicio, por lo que la forma más fácil de llegar a un n?...

El principal objetivo terapéutico en desprendimiento de retina (DR) es encontrar una manera de limitar el daño celular y la inflamación de la retina fotorreceptor resultante de la separación de los fotorreceptores de las células epiteliales pigmentadas de la retina. Durante RD, las células de RPE se activan, migran, dedifferentiate, y proliferan en la superficie de la retina desprendida, ejerciendo fuerzas contráctiles que conducen a complicaciones. Análisis Transcriptómica de RD es una manera de identificar los genes diana con la expresión modificada como consecuencia de RD y por lo tanto las futuras moléculas terapéuticas que podrían mejorar el resultado visual final en combinación con la cirugía. Es bien sabido ácido ribonucleico (ARN) no es estable como es el ácido desoxirribonucleico (ADN), este último siendo utilizado ampliamente para estudios genéticos, su estabilidad había permitido la secuenciación del genoma del Neandertal a partir de muestras paleontológicos de más de 30.000 años de edad 2. La transcripción de ADN de los genes del genoma en ARN mensajero es laimportante proceso en la expresión génica, y el ARNm es lábil con el fin de constituir una señal. RNAs son rápidamente degradados por las enzimas ARNasa que terminan la señal. Cuando los tejidos se aíslan a partir de un organismo, los ARN son muy a menudo degrada antes de la expresión se estudió en un laboratorio de investigación. ARN degradados no es adecuado para el análisis de la expresión génica. Debido a que el personal de laboratorio no puede participar en la cirugía, se desarrolló un procedimiento que es fácil y sólo requiere que el cirujano para recuperar los tejidos en una solución de RNasa libre apropiado. Los ARN de los tejidos es estable y pueden ser analizados sin ningún signo de degradación después de 72 horas a temperatura ambiente en esta solución. Los ARN a partir de muestras se purifican por un método normalizado que implica una ultracentrifugación en un gradiente de cloruro de cesio después de haber sido transferidos al laboratorio 3. Entonces, la calidad de los ARN se evaluó mediante electroforesis en gel de agarosa y por RT-PCR. El protocolo de purificación de ARN tiene laventaja de separar las moléculas de acuerdo con su densidad que es diferente para el ADN y el ARN y asegura que el ARN no está contaminado por unas moléculas de ADN que generarán señales de artefactos en estudios de expresión génica. Además, los ARN de transferencia (ARNt), que son cuantitativamente los ARN más abundantes dentro de una célula, se separan de acuerdo con la misma propiedad física a partir del ARN ribosómico (ARNr) y los ARN mensajeros (ARNm), estos dos últimos uno de ellos el el producto final del proceso de purificación. La eliminación de ARNt de la preparación es útil ya que la mayoría de los protocolos de análisis de microarrays implicado el uso de transcriptasas inversas y polimerasas de ARN que son inhibidas por el ARNt 4-6. El ARN purificado a partir de muestras quirúrgicas están etiquetadas utilizando el protocolo estándar y se hibridan a un chip de micromatriz y los resultados se analizaron usando dos métodos complementarios, el método de la tasa de falso descubrimiento, y el uso de un nuevo método basado en la información mutua y visualized en el servidor basado en web Retinobase 7,8.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Aventi J-E</td>
 <td></td>
 </tr>
 <tr>
 <td>Rotor</td>
 <td>Beckman Coulter</td>
 <td>JS-5.3</td>
 <td></td>
 </tr>
 <tr>
 <td>Ultracentrifuge</td>
 <td>Beckman Coulter</td>
 <td>LE 80K</td>
 <td></td>
 </tr>
 <tr>
 <td>Rotor</td>
 <td>Beckman Coulter</td>
 <td>SW41</td>
 <td></td>
 </tr>
 <tr>
 <td>Power supply</td>
 <td>Biorad</td>
 <td>Pac 3000</td>
 <td></td>
 </tr>
 <tr>
 <td>PolytronTM</td>
 <td>Kinematica</td>
 <td>PT 2100</td>
 <td>Supplied with PT-DA 2105:2</td>
 </tr>
 <tr>
 <td>Agarose gel electrophoresis device</td>
 <td>Biorad</td>
 <td>MiniGel Cell GT</td>
 <td></td>
 </tr>
 <tr>
 <td>Imaging System </td>
 <td>Biorad</td>
 <td>GelDoc-It Imager</td>
 <td></td>
 </tr>
 <tr>
 <td>5 ml sterile polyethylene tube</td>
 <td>Greiner</td>
 <td>115261</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile polyallomer centrifuge tube </td>
 <td>Beckman Coulter</td>
 <td>331372</td>
 <td></td>
 </tr>
 <tr>
 <td>Chemical reagents</td>
 <td>Sigma</td>
 <td></td>
 <td>Molecular biology grade (RNase and DNase free products)</td>
 </tr>
 <tr>
 <td>Cleaning Solution </td>
 <td>VWR </td>
 <td>RBS</td>
 <td></td>
 </tr>
 <tr>
 <td>Microarray chip</td>
 <td>Affymetrix</td>
 <td>Human U133 plus 2 array</td>
 <td></td>
 </tr>
 </tbody>
</table>

transcriptomic analysis of human retinal surgical specimens using journal

El desprendimiento de retina (DR) describe una separación de la retina neurosensorial del epitelio pigmentario de la retina (RPE). El RPE es esencial para la función normal de las neuronas sensibles a la luz, los fotorreceptores. Desprendimiento de la retina de la RPE crea una brecha física que se llena con el fluido extracelular. RD inicia los eventos adversos celulares y moleculares que afectan tanto a la retina neurosensorial y el EPR ya que el intercambio fisiológico de iones y metabolitos se ve gravemente perturbado. La consecuencia para la visión está relacionada con la duración de la separación desde una rápida reapposition de los dos tejidos en los resultados de la restauración de la visión 1. El tratamiento de la RD es exclusivamente quirúrgico. La eliminación del gel vítreo (vitrectomía) es seguido por la eliminación no parte esencial de la retina alrededor de la zona individual para favorecer el desprendimiento de retina. Las muestras de retina retirados son res nullius (nada) y por lo tanto normalmente se desechan.Para recuperar el ARN de estas piezas quirúrgicas, desarrollamos el Diario procedimiento que permite la conservación de ARN durante la transferencia del bloque quirúrgico para el laboratorio. También estandarizado un protocolo para purificar el ARN mediante ultracentrifugación cloruro de cesio para asegurar que los ARN purificados son adecuados para el análisis de la expresión génica global. La calidad del ARN fue validado tanto por RT-PCR y análisis de microarrays. El análisis de los datos muestra una participación simultánea de la inflamación y degeneración de los fotorreceptores durante RD.

El procedimiento de auditoría (Figura 1) permite recuperar muestras de retina del bloque quirúrgico para ARN purificado, y para analizar el transcriptoma de desprendimiento de retina. Resultados de desprendimiento de retina en la regulación al alza de la quimiotáctica de monocitos MCP1 gen CCL2, y en la disminución de la expresión de la varilla fotorreceptor transducir gen GNAT1, el cono de onda corta opsina OPN1SW, y la homeogene CRX (Figura 2).</strong...

Watch this Scientific Journal Video about Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl at JoVE.com

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Utilizamos muestras de la retina de retinectomía para un análisis transcriptomic de desprendimiento de retina. Hemos desarrollado un procedimiento que permite la conservación de RNA entre los bloques quirúrgicos y el laboratorio. Se estandarizó un protocolo para purificar el ARN mediante ultracentrifugación en cloruro de cesio para asegurar que los ARN purificados son adecuados para el análisis de microarrays.

Análisis Transcriptomic de especímenes quirúrgicos retina humana usando Diario

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

transcriptomic-analysis-human-retinal-surgical-specimens-using

Research

JoVE Journal

Medicine

7.7K vistas.  Institut National de la Santé et de la Recherche Médicale. Utilizamos muestras de la retina de retinectomía para un análisis transcriptomic de desprendimiento de retina. Hemos desarrollado un procedimiento que permite la conservación de RNA entre los bloques quirúrgicos y el laboratorio. Se estandarizó un protocolo para purificar el ARN mediante ultracentrifugación en cloruro de cesio para asegurar que los ARN purificados son adecuados para el análisis de microarrays.

Video: Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Método para el desarrollo de nuevos fármacos contra el cáncer utilizando explantes tumorales de las piezas quirúrgicas


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Investigación

Medicina

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Protocolo mejorado para microdisección láser de islotes pancreáticos humanos a partir de muestras quirúrgicas

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Dos métodos para fijar las células humanas primarias endometrial estromales de la histerectomía especímenes

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Un Paso a Paso Protocolo para la cirugía subretiniana en conejos

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

Generación de un ratón humanizado hígado uso de células madre hepáticas humanas

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

La microscopia intravital de la utilización de la vasculatura asociada a Tumor avanzado Dorsal cámaras de ventana pliegues cutáneos en ratones transgénicos fluorescentes

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Usando In Vitro células vivas imágenes para explorar quimioterápicos entregado por nanopartículas basadas en lípidos

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. Recent technological progress has enabled the fabrication of microelectrode arrays that allow recording the activity of multiple individual neurons in the neocortex of the human brain. These arrays have the potential to bring unique insight onto the neuronal correlates of cerebral function in health and disease. Furthermore, the identification and decoding of volitional neuronal activity opens the possibility to establish brain-computer interfaces, and thus might help restore lost neurological functions. The implantation of neocortical microelectrode arrays is an invasive procedure requiring a supra-centimetric craniotomy and the exposure of the cortical surface; thus, the procedure must be performed by an adequately trained neurosurgeon. In order to provide an opportunity for surgical training, we designed a procedure based on a human cadaver model. The use of a formaldehyde-fixed human cadaver bypasses the practical, ethical and financial difficulties of surgical practice on animals (especially non-human primates) while preserving the macroscopic structure of the head, skull, meninges and cerebral surface and allowing realistic, operating room-like positioning and instrumentation. Furthermore, the use of a human cadaver is closer to clinical daily practice than any non-human model. The major drawbacks of the cadaveric simulation are the absence of cerebral pulsation and of blood and cerebrospinal fluid circulation. We suggest that a formaldehyde-fixed human cadaver model is an adequate, practical and cost-effective approach to ensure proper surgical training before implanting microelectrode arrays in the living human neocortex.

We designed a procedure in which a formaldehyde-fixed human cadaver is used to assist neurosurgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain.

Formación quirúrgica para la implantación de microelectrodos Neocortical matrices usando un modelo de cadáver humano formaldehído-fija

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. ...

Diagnosis and Surgical Treatment of Human Brucellar Spondylodiscitis

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in pastoral communities. It is difficult to differentially diagnose and usually leads to irreversible neurologic deficits and spinal deformities. The initial diagnosis of BS is based on clinical findings and radiographic assessments, and the confirmed diagnosis should be established by the isolation of Brucella species from the blood and/or the standard tube agglutination test. Differential diagnosis of multifocal BS from either degenerative disc diseases or tuberculosis is especially highlighted. The surgical approach, either endoscopic or open, is demonstrated in detail, accompanied by radiographic evidence of structural compression or severe instability. Further, the crucial surgical steps, including single-stage transforaminal decompression, debridement, interbody fusion, and internal fixation, are explained. Moreover, perioperative care and postoperative rehabilitation are also addressed. Taken together, this clinical algorithm presents a practical guide that has yielded substantially satisfactory outcomes in the past decades, which can also be introduced for large-scale application to manage human BS, especially in endemic regions.

We describe a clinical algorithm, based on decades of front-line experience of diagnosis and surgical treatment of human Brucellar spondylodiscitis in the largest medical center of the China's Xinjiang Pastoral Area.

Diagnóstico y Tratamiento Quirúrgico de la Espondylodiscitis Humana Brucellar

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Inducción y análisis del estrés oxidativo en células epiteliales pigmentarias de la retina humana transfectadas por transposones de la bella durmiente

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...