Agradecemos Jianhua Ling, Raymond MacDonald, Galvin Swift, Michelle Griffin, Paul Grippo y Satyanarayana Rachagani por sus útiles comentarios, sugerencias y compartir sus protocolos. Este trabajo fue apoyado por U01CA111294 subvención y un premio de desarrollo de la idea del Programa de Investigación Intramural la Universidad Estatal de Ohio.

1. Preparación Las siguientes prácticas se adhieren a las políticas establecidas por el Cuidado de Animales y el empleo Comisión de la Institución (IACUC). <ol><li> Se recomienda aislar a no más de 6 páncreas de ratón en un día determinado. Tener todos los instrumentos quirúrgicos limpios y esterilizados en autoclave, un juego por animal. </li><li> Marque todos los tubos de microcentrífuga. Cuatro tubos de 2 ml por animal. </li><li> Coloque 8 ml de reactivo de lisis en 50 ml tubos de centrífuga en hielo. Nota: Mientras que el kit de purificación viene con un reactivo de fenol de tiocianato de guanidina que es adecuado para el aislamiento páncreas, reactivo de lisis se utiliza debido a los grandes volúmenes que son necesarios por el aislamiento. </li><li> Vierta aproximadamente 10 ml de las siguientes soluciones en 15 ml tubos de centrífuga para la limpieza de las cuchillas del homogeneizador. Etanol, 70% (2 tubos), agua de calidad HPLC (1 tubo) y agua de grado HPLC que contiene aproximadamente 200 l de RNasa visitante o inactivador ribonucleasa equivalente. </li></ol&gt; 2. Cirugía, Dissection Páncreas y homogeneización <ol><li> Coloque 1.2 ml de isoflurano en un frasco que contiene gasa de algodón o toallas de papel. Los ratones se colocan en una plataforma que les impide alcanzar la fuente de la isoflurano. Añadir isoflurano fresca en la jarra antes de anestesiar cada ratón. </li><li> Con cuidado, coloque el ratón en el frasco que contiene el isoflurano. Tapar el frasco y controlar los efectos de la anestesia, girando suavemente el frasco de un lado a otro. El animal se anestesia una vez que está en incapaz de soportar en su &#39;propia. </li><li> Para la confirmación de la anestesia, retire al animal del frasco y aplicar una pizca dedo del pie firme. Si el ratón no reacciona a la pizca dedo del pie, siga en el paso 2.5. </li><li> Si el ratón reacciona a la pizca dedo del pie, a continuación, repita los pasos 2.2 hasta 2.3. </li><li> Colocar el animal anestesiado en la mesa de trabajo en la posición prona. Dislocar el cuello uterino de ratón por fuerza colocando la mano izquierda sobre el cuello uterino de ratón. Nosotrosción de la mano derecha agarre la cola del ratón y tire hacia arriba de forma rápida a un ángulo aproximado de 45 ° para dislocar el cuello del útero. </li><li> Fije la carcasa a una superficie (una pieza nivel de espuma de poliestireno cubierta con toallas de papel) por la perforación de cada uno de cuatro patas de los animales en la superficie plana usando calibre 25 agujas hipodérmicas. </li><li> El uso de instrumentos quirúrgicos estériles, cortar la sección media del ratón abierta y rápidamente extirpar el páncreas del ratón. Utilice las pinzas para sujetar el páncreas y cortarlo del bazo y el intestino delgado utilizando tijeras de primavera. Tenga cuidado de no estirar el páncreas durante la eliminación del uso del ratón. La alteración del tejido podría liberar ribonucleasa. Nota: El tiempo que se necesita para extirpar el páncreas del ratón es crítica. Un experto en la materia debe ser capaz de diseccionar el páncreas en aproximadamente 1 min o menos. </li><li> Coloque todo el páncreas en un tubo de 50 ml que contiene 8 ml de hielo reactivo de lisis frío. Tapar el tubo y agitar brevemente para sumergir el tiN ú mero en el reactivo y proceder a la siguiente etapa sin demora. La proporción de reactivo de lisis a los tejidos es crítico. Ver la sección de resultados para una comparación de la integridad del ARN aislado usando diferentes volúmenes de reactivo de lisis. </li><li> Homogeneizar durante 5 seg. Es importante presionar las cuchillas homogeneizador para la parte inferior del tubo de centrífuga para permitir que el tejido sea arrastrado hacia las cuchillas para la homogeneización eficiente. </li></ol> 3. Eliminación de tejido no digerido Nota: Después de la homogeneización, pequeños trozos de tejido se mantendrán en el reactivo de lisis. Es más importante para lisar rápidamente el tejido dejando algo de tejido sin digerir en lugar de sobre la homogeneización y la degradación riesgo de ARN. <ol><li> Transferencia de 2 ml de reactivo de lisis que contiene el páncreas lisadas a un tubo de microcentrífuga de 2 ml. Recoge dos tubos de microcentrífuga de homogeneizado por el aislamiento. Deseche el material homogeneizado restante o almacenar a -80 ° C para futuros estudios. </li><li> Centavorifuge a 4 º C, 12000 × g durante 5 min para sedimentar el tejido no digerido. Este es un paso crítico como el arrastre de tejido no digerido en soluciones posteriores de ARN probablemente contamine la muestra con ribonucleasa (paso 5,13). </li><li> Transferencia de 1 ml del sobrenadante en un tubo de microcentrífuga de 2 ml. Proceda con el aislamiento inmediato y guarde el tubo de réplica a -80 ° C para su uso futuro. Coloque todos los tubos que contienen homogeneizado en hielo húmedo mientras se procede al siguiente paso. </li><li> Deseche el reactivo de lisis restante en un recipiente para desechos químicos apropiados. </li></ol> 4. Limpieza de las cuchillas Homogeneizador <ol><li> Antes de proceder a la siguiente aislamiento, limpiar las cuchillas homogeneizador colocándolos en 10 ml de etanol al 70%. Active el homogeneizador por alrededor de 20 segundos para quitar cualquier pedazo de tejido que pueden estar atrapados en las cuchillas. </li><li> Utilice una punta de pipeta de 200 l para eliminar las piezas que permanecen en las cuchillas homogeneizador. </li&gt; <li> Repita el paso 4.1 en el agua, en general inactivador Rnase y etanol al 70%. Secar el eje homogeneizador con una toallita limpia Kim. </li><li> Repita el paso 2.1 para el aislamiento del páncreas de los animales restantes, manteniendo ARN aislado de cada animal por separado en lugar de puesta en común de la ARN. </li></ol> 5. Aislamiento de ARN La siguiente sección es idéntica a el protocolo del kit de purificación. La ventaja de el kit de purificación es que va a aislar el ARN total, incluyendo pequeños RNAs. <ol><li> Si se utiliza la solución homogeneizado congelado, descongelar en hielo húmedo y dejar reposar a temperatura ambiente durante 5 min. </li><li> Añadir 200 l de cloroformo, tapar el tubo y agitar enérgicamente a mano durante 15 segundos. Deje reposar el tubo a temperatura ambiente durante 2-3 min. </li><li> Centrifugar durante 15 minutos a 12.000 xg, 4 º C </li><li> Transferir la capa acuosa superior a un tubo de microcentrífuga. Añadir 1,5 volúmenes de isopropanol y mezclar bien con la pipeta hacia arriba y abajo varias veces. </li><li> Traslado hasta el 700 l de la muestra en una columna de centrifugación que se coloca en un tubo de recogida de 2 ml. Cierre la tapa y centrifugar a 8000 xg durante 15 segundos a temperatura ambiente. </li><li> Deseche el flujo directo. </li><li> Repetir los pasos 5.5 a 5.6, utilizando el resto de la muestra. </li><li> Añadir 700 l Buffer TAR a la columna de centrifugación. Centrifugar durante 15 segundos a 8000 x g. Deseche el flujo directo. </li><li> Pipetear 500 l de tampón RPE en la columna de centrifugación. Centrifugar durante 15 segundos a 8.000 x g para lavar la columna. Deseche el flujo directo. </li><li> Añadir 500 l de tampón RPE a la columna de centrifugación. Centrifugar durante 2 min a 8000 xg para secar la columna. </li><li> Transferir la columna de centrifugación a un tubo de microcentrífuga de 1,5 ml. Pipetear 80 l de agua libre de ribonucleasa directamente sobre la membrana de la columna de centrifugación. Centrifugar durante 1 min a 8000 xg para eluir el ARN. </li><li> Añadir 1 l inhibidor de RNasa a la solución de ARN. Almacenar a -80 ° C o proceder a 5.13. </li><li> Para validar la integridad del ARN, primero calcular la conce ARNntration y 260/280 escala utilizando un espectrofotómetro. </li><li> Diluir una porción de la solución de ARN a aproximadamente 500 ng / l con agua de grado de biología molecular. </li><li> Determinar el ARN integridad (es decir, RIN) utilizando el análisis de electroforesis capilar. Aproximadamente 5 l de solución de ARN es suficiente para el análisis. </li><li> Alícuota de la solución de ARN (véase la Figura 2) y se almacena a -80 ° C. </li></ol>

<ol>
	<li>Chen, C., Ridzon, D. A., Broomer, A. J., Zhou, Z., Lee, D. H., Nguyen, J. T., Barbisin, M., Xu, N. L., Mahuvakar, V. R., Andersen, M. 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=Real-time+quantification+of+microRNAs+by+stem-loop+RT-PCR.">Real-time quantification of microRNAs by stem-loop RT-PCR.</a> Nucleic Acids Res. 33, e179 (2005).</li><li>Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., Rutter, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+biologically+active+ribonucleic+acid+from+sources+enriched+in+ribonuclease.">Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.</a> Biochemistry. 18, 5294-5299 (1979).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction.">Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.</a> Anal Biochem. 162, 156-159 (1987).</li><li>Griffin, M., Abu-El-Haija, M., Abu-El-Haija, M., Rokhlina, T., Uc, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+and+versatile+method+for+isolation+of+high-quality+RNA+from+pancreas.">Simplified and versatile method for isolation of high-quality RNA from pancreas.</a> Biotechniques. 52, 332-334 (2012).</li><li>Iyer, V. R., Eisen, M. B., Ross, D. T., Schuler, G., Moore, T., Lee, J. C., Trent, J. M., Staudt, L. M., Hudson Jr, J., Boguski, M. S., 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+transcriptional+program+in+the+response+of+human+fibroblasts+to+serum.">The transcriptional program in the response of human fibroblasts to serum.</a> Science. 283, 83-87 (1999).</li><li>Lenstra, J. A., Beintema, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amino+acid+sequence+of+mouse+pancreatic+ribonuclease.+Extremely+rapid+evolutionary+rates+of+the+myomorph+rodent+ribonucleases.">The amino acid sequence of mouse pancreatic ribonuclease. Extremely rapid evolutionary rates of the myomorph rodent ribonucleases.</a> European journal of biochemistry FEBS. 98, 399-408 (1979).</li><li>Mullin, A. E., Soukatcheva, G., Verchere, C. B., Chantler, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+in+situ+ductal+perfusion+to+facilitate+isolation+of+high-quality+RNA+from+mouse+pancreas.">Application of in situ ductal perfusion to facilitate isolation of high-quality RNA from mouse pancreas.</a> Biotechniques. 40, 617-621 (2006).</li><li>Rio, D. C., Ares, M., Hannon, G. J., Nilsen, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RNA A Laboratory Manual. , (2011).</li><li>Streit, S., Michalski, C. W., Erkan, M., Kleeff, J., Friess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Northern+blot+analysis+for+detection+and+quantification+of+RNA+in+pancreatic+cancer+cells+and+tissues.">Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues.</a> Nat Protoc. 4, 37-43 (2009).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> Journal of Visualized Experiments JoVE. , (2011).</li></ol>

The authors have nothing to disclose.

Aislar ARN de tejidos que se ribonucleasa rico representa un gran reto para los experimentos de biología molecular. Varios reactivos y kits están disponibles en el mercado que se basan principalmente en el método de tiocianato de guanidina fenol de la extracción de RNA. Tiocianato de guanidina desnaturaliza la proteína y por lo tanto reduce la actividad de la ribonucleasa. Sin embargo, la magnitud de la ribonucleasa en el páncreas requiere modificaciones adicionales a los protocolos estándar de aislamiento de ARN...

Se requiere Aislamiento de ARN con una alta integridad para los experimentos de biología molecular de rutina, como el norte de borrones 9, QRT-PCR 1 o perfiles de expresión genética 5. La mayoría de los métodos contemporáneos de aislamiento de ARN se basan en modificaciones de los protocolos de tiocianato de guanidina 2, 3. Tiocianato de guanidina es un fuerte desnaturalizante de proteínas y se interrumpir efectivamente la actividad de la ribonucleasa bajo la mayoría de condiciones. El método popular de Chomczynski y Sacchi 3 combinó fenol a la solución de tiocianato de guanidina de lisis, lo que reduce el tiempo de aislamiento a aproximadamente 4 horas. Muchos reactivos de extracción de ARN comercialmente disponibles se basan en el método de Chomczynski y Sacchi 3. Ribonucleasa tejidos ricos como humano o de ratón páncreas presenta un desafío adicional para el aislamiento de ARN. Ratón páncreas puede contener hasta 75 mg de ribonucleasa 6 algunos de los cuales se dará a conocer During interrupción del tejido pancreático que resulta en la autolisis de los tejidos. Las modificaciones de los protocolos de tiocianato de guanidina se han utilizado con éxito para aislar ARN a partir de páncreas con alta integridad 2, 4, 7, 10. Infusión de ARN de estabilización reactivo en páncreas de ratón aislamiento facilitado de ARN con alta integridad 4, 7, 10, sin embargo infusión de soluciones en el páncreas requiere una gran habilidad, pueden requerir instrumentos especializados tales como un microscopio de disección y alterar la arquitectura del tejido durante el procedimiento puede resultar en la lisis celular. Perfusión tisular con el reactivo de estabilización de ARN podría interferir con otras aplicaciones como el aislamiento de proteínas y la tinción histológica. Además, esta técnica no es adecuada para el aislamiento de ARN a partir de páncreas humano. Otros protocolos requieren la preparación de soluciones específicas del investigador 2. Se presenta un protocolo para aislar ARN con una alta integridad de páncreas de ratón. Thprotocolo de correo utiliza elementos de los métodos publicados anteriormente y se basa en gran parte de los protocolos de reactivo de lisis estándar de fenol / guanidina basado en tiocianato. No requiere la preparación de soluciones especializados ni requiere la infusión de los reactivos en el páncreas. Los pasos críticos para el aislamiento de ARN éxito incluyen un fenol alta / a base de guanidina lisis reactivo proporción de tejidos, extracción de tejido no digerido antes de la separación de fases y la inclusión de un inhibidor de ribonucleasa a la solución de ARN resultante. El uso de estas y otras modificaciones, que típicamente aislar el ARN con Rin mayor que 7 para ser utilizado para el análisis de la expresión génica de rutina.

<table border="0" cellpadding="0" cellspacing="0" width="788">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:93px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:360px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Power Gen 500&#160;</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">14-261-03&#160;</td>
			<td>Homogenizer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5424R</td>
			<td style="width:93px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td>Refrigerated microcentifuge</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trizol reagent</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;">15596018</td>
			<td>Lysis reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Student Vannas spring scissors</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">91500-09&#160;</td>
			<td>use to cut pancreas from attached tissues</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical scissors, 6 inch</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">08-951-20</td>
			<td>use to cut scin of mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt end forceps</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">1381239</td>
			<td>use to hold pancreas while dissecting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isoflurane USP</td>
			<td style="width:93px;">Abbott Labs</td>
			<td style="width:119px;">4/8/5210</td>
			<td>Anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rnase out (40 U/&#181;l)</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;">10777-019</td>
			<td>Rnase inhbitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Rnase Away</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">10328-011</td>
			<td>General Rnase inactivator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC grade water</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">W5-4</td>
			<td>For washing homogenizer blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Molecular Biology grade water</td>
			<td style="width:93px;">Hyclone</td>
			<td style="width:119px;">SH30538.03</td>
			<td>Elution of RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">miRNeasy Mini kit</td>
			<td style="width:93px;">Qiagen</td>
			<td style="width:119px;">217004</td>
			<td>Purification Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2 ml microcentrifuge tubes, certified Rnase Dnase free</td>
			<td style="width:93px;">USA Scientific Plastics</td>
			<td style="width:119px;">1620-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml centrifuge tubes</td>
			<td style="width:93px;">Falcon</td>
			<td style="width:119px;">352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% ethanol</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100% ethanol</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">200 ul pipet tips</td>
			<td style="width:93px;">Rainin</td>
			<td style="width:119px;">GPL200F</td>
			<td>For cleaning homogenizer blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">BP1145-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nanodrop</td>
			<td style="width:93px;">Thermo</td>
			<td style="width:119px;">ND-1000</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bioanalyzer</td>
			<td style="width:93px;">Agilent</td>
			<td style="width:119px;"></td>
			<td>Capillary electrophoresis analyzer to measue RNA integrity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNAlater</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;"></td>
			<td>RNA Stabilization Reagent&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNeasy&#160;spin columns&#160;</td>
			<td style="width:93px;">Qiagen</td>
			<td style="width:119px;"></td>
			<td>&#160;spin columns&#160;as a part of the miRNeasy Mini kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mice</td>
			<td style="width:93px;">Chales River</td>
			<td style="width:119px;">strain C57BL/6</td>
			<td style="width:360px;">Their age ranged from 4 to 12 months.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mice&#160;</td>
			<td style="width:93px;">Jackson Lab</td>
			<td style="width:119px;">strain 129</td>
			<td style="width:360px;">Their age ranged from 4 to 12 months.&#160;&#160;</td>
		</tr>
	</tbody>
</table>

rna isolation from mouse pancreas: a ribonuclease-rich tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

El rendimiento total de ARN a partir de 1 ml de homogeneizado de lisis es 20-40 mg. OD 260/280 ratios son típicamente alrededor de 2.0 y el RIN son consistentemente superior a 7,0. Si el RIN es ≤ 6, será necesario repetir el aislamiento. De vez en cuando, se consigue un RIN que es mayor que 8,0. Los dos métodos más utilizados de la eutanasia de los ratones antes de la extracción del páncreas son asfixia de CO 2 o la inhalación de isoflurano. Ambas técnicas son seguidos p...

Watch this Scientific Journal Video about RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue at JoVE.com

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Aislamiento de ARN de ratón Páncreas: Un tejido de ribonucleasa-rica

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

rna-isolation-from-mouse-pancreas-a-ribonuclease-rich-tissue

Research

JoVE Journal

Biology

27.3K vistas.  The Ohio State University. We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Video: RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

Aislamiento de Miofibroblastos primarias de ratón y el tejido del colon humano

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Investigación

Biología

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

In Vitro Páncreas Organogenesis de dispersos embrionarios de ratón Progenitores

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Proteína Aislamiento del desarrollo embrionario del corazón del ratón Válvula Región

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Aislamiento y análisis funcional de las mitocondrias de las células cultivadas y ratón de Tejidos

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

Aislamiento de núcleos de tejido entero utilizando un método de detergente y libre de enzimas

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Recolección de tejidos y extracción de ARN de la articulación de la rodilla osteoartrítica humana

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

Aislamiento de núcleos de riñón de ratón adulto para secuenciación de ARN de un solo núcleo

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria

Plastoglobule lipid droplets are a dynamic sub-compartment of plant chloroplasts and cyanobacteria. Found ubiquitously among photosynthetic species, they are believed to serve a central role in the adaptation and remodeling of the thylakoid membrane under rapidly changing environmental conditions. The capacity to isolate plastoglobules of high purity has greatly facilitated their study through proteomic, lipidomic, and other methodologies. With plastoglobules of high purity and yield, it is possible to investigate their lipid and protein composition, enzymatic activity, and protein topology, among other possible molecular characteristics. This article presents a rapid and effective protocol for the isolation of plastoglobules from chloroplasts of plant leaf tissue and presents methodological variations for the isolation of plastoglobules and related lipid droplet structures from maize leaves, the desiccated leaf tissue of the resurrection plant, Eragrostis nindensis, and the cyanobacterium, Synechocystis sp. PCC 6803. Isolation relies on the low density of these lipid-rich particles, which facilitates their purification by sucrose density flotation. This methodology will prove valuable in the study of plastoglobules from diverse species.

A fast and efficient protocol is presented for the isolation of plastoglobule lipid droplets associated with various photosynthetic organisms. The successful preparation of isolated plastoglobules is a crucial first step that precedes detailed molecular investigations such as proteomic and lipidomic analyses.

Aislamiento de gotitas lipídicas de plastoglobulo del tejido foliar de plantas y cianobacterias

Plastoglobule lipid droplets are a dynamic sub-compartment of plant chloroplasts and cyanobacteria. Found ubiquitously among photosynthetic species, they ...

Cultivo primario de células madre derivadas del tejido adiposo del epidídimo de ratón

Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential to modulate the immune response. The MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients based on low expression of class I major histocompatibility complex (MHC) molecules and in the use of cell-based therapy for allogeneic transplant. These cells can be isolated from several tissues, the most commonly used being the bone marrow and adipose tissues. We provide an easy protocol to isolate, culture, and characterize MSCs from epididymal adipose tissue of mice. The epididymal adipose tissue is surgically excised, physically fragmented, and digested with 0.15% collagenase type II solution. Then, primary adipose tissue-derived stem (ADSCs) cells are cultured and expanded in vitro, and the phenotypic characterization is performed by flow cytometry. We also provide the steps to differentiate the ADSCs into osteogenic, adipogenic, and chondrogenic cells, followed by functional characterization of each cell lineage. The protocol provided here can be used for in vivo and ex vivo experiments, and as an alternative, the adipose-derived stem cells can be used to generate MSCs-like immortalized cells.

Autor destacado: Avance de los enfoques de tratamiento con células madre derivadas del tejido adiposo

The adipose tissue is an excellent source of mesenchymal stem cells. Here, we bring the step-by-step extraction, cultivation, and characterization of adipose tissue-derived stem cells (ADSCs) from Swiss mice epididymal adipose tissue.

Aislamiento, expansión in vitro y caracterización de células madre mesenquimales a partir de tejido adiposo epididimario de ratón

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential ...