We thank Dr. Vicky Macias, Angeline Giangreco and Avani Vaishnav for assistance with optimizing this methodology over the years and Yang Zhang and Dr. Jian Ma at the University of Illinois at Urbana-Champaign for the RNA seq analysis. This work was supported by NIH/NCI R01 CA166588-01 (Nonn), American Cancer Society Research Scholar 124264-RSG-13-012-01-CNE (Nonn), NIH/NCI R03 CA172827-01 (Nonn), DOD-CDMPR PRCP Health Disparities Idea Award PC121923 (Nonn) and a Prevent Cancer Foundation grant (Zhou).

Todos los tejidos humanos utilizados para estos experimentos fueron adquiridas a través de un protocolo aprobado la Junta de Revisión Institucional y / o exención de la Universidad de Illinois en Chicago. 1. Sección fresco congelado de próstata en PEN-diapositiva y en Charged portaobjetos de vidrio <ol><li> El día anterior o unas horas antes de seccionar la muestra, limpie las herramientas (es decir, cepillo, fórceps, coplins, cuchillas, diapositivas enmarcadas-PEN (si no está ya RNasa libre), un marcador de ETOH seguro y un lápiz) y el interior de un criostato RT con una solución de RNasa descontaminar utilizando una botella de spray y de laboratorio-toallitas. <ol><li> Deje que la solución de RNasa descontaminar reposar por 5 minutos antes de limpiar de distancia, enjuague con DEPCH 2 0 para eliminar sobrantes solución y un enjuague final con EtOH al 70% (preparado con DEPCH 2 0) RNasa descontaminación. Nota: Es esencial para mantener un espacio de trabajo libre de RNasa. Todos coplins, herramientas, medios de montaje congelado, cuchillas y diapositivas utilizadas deben ser utilizados sólo para RNasa trabajo librey nunca se utiliza en un ambiente libre de norma no RNasa. Todos los instrumentos deben ser tratados con una solución de descontaminación RNasa antes de cada experimento. </li></ol></li><li> Enfriar criostato a -24 ° C. </li><li> Coloque los siguientes instrumentos gratuitas RNasa limpiadas dentro criostato para enfriar al menos 30 minutos antes de seccionar: Diapositiva Coplin llena con etanol al 100%, las herramientas, las pequeñas cintas de tejido congelado y el mandril de montaje. </li><li> Recuperar el tejido prostático congelado de almacenamiento criogénico y el lugar en un cubo de espuma lleno de hielo seco para el transporte. Coloque tejido en criostato equilibrar a criostato temperatura durante al menos 30 min. Nota: Los tejidos de la próstata humanos para láser captura micro-disección pueden ser fresco congelado o fijado en formol e incluido en parafina (FFPE). Las limitaciones de tiempo y la preparación de diapositivas son ligeramente diferentes entre las fuentes de tejido. Aquí se describen los pasos para secciones congeladas. </li><li> Trabajar con un tejido a la vez, y vierta medio de montaje congelado en el Bottom de la casete y rápidamente montar el tejido de la próstata congelado en el medio de montaje congelado usando las pinzas refrigeradas. </li><li> Coloque el casete con el tejido en enfriadores en criostato durante 5 min para establecer medio de montaje congelado. </li><li> Aplique una pequeña cantidad de medio de montaje congelado en el plato y presione con cuidado el lado inferior del tejido montada arriba en el medio de montaje congelado. Deje reposar en el criostato durante 5 min para permitir que el medio de montaje congelado se solidifique por completo. </li><li> Coloque tirada en el soporte y apriete. </li><li> Bloquear el mango en la posición de bloqueo y coloque una nueva cuchilla en el criostato. Para los puntos finales moleculares aguas abajo que incluyen una etapa de amplificación (es decir, qPCR), es aconsejable utilizar una cuchilla nueva para cada paciente para evitar la contaminación (o mover la hoja de más de utilizar el área de la cuchilla para la siguiente muestra). </li><li> Desbloquear el mango y avanzar cuidadosamente el tejido a mano o con un pedal de pie para igualar y exponer el tejido con 50 μm secciones hasta que la exposición de tejido deseado es alcanzado. </li><li> Ajuste criostato a 5 micras, cortar una o dos secciones y colocarlos en portaobjetos de vidrio cobrado por hematoxilina y eosina (H &amp; E) tinción (ver paso 2). </li><li> Coloque inmediatamente la diapositiva en el etanol frío al 100% durante 2 min. </li><li> Retire la lámina de vidrio cargada de etanol y dejar secar a temperatura ambiente antes de proceder a la tinción de H &amp; E (Paso 2). </li><li> Coloque RT diapositivas PEN-marco en un partidario marco. </li><li> Ajuste criostato a 10 secciones micras y lugar en las diapositivas de marco RT PEN. Uno a cuatro secciones pueden ser lugar en cada diapositiva dependiendo del tamaño del tejido. </li><li> Sumergirse inmediatamente la diapositiva en el etanol frío al 100% durante 2 min. </li><li> Retire las diapositivas PEN-frame a partir de etanol y almacenar el portaobjetos en una caja de portaobjetos en una caja en un desecador de -80 ° C congelador hasta el momento de LCM. Tres días es el tiempo máximo de almacenamiento para una recuperación óptima de ARN. </li><li> Continúe con el Paso 2 por sólo el vidrio cargada deslizómi. Continúe con el Paso 3 para diapositivas PEN-marco. </li></ol> 2. hematoxilina y eosina-Y (H &amp; E) de la mancha para histológico Mark-up Nota: Esto si por el tejido en el portaobjetos de vidrio cargada y no la diapositiva PEN-marco para LCM. <ol><li> Hidratar las secciones sobre el portaobjetos de vidrio cargada de inmersión la corredera a través de etanol clasificado como sigue: sumergir el portaobjetos dos veces en etanol al 100% durante 3 min, dos veces en etanol al 95% durante 3 min, una vez en etanol al 70% durante 3 min y dos veces en H2O destilada durante 3 min. <ol><li> Preparar la solución de alcohol de ácido: 99 ml 70% de etanol + 1 ml 1% de HCl y una solución de bicarbonato de sodio 0,1%: 0,1 g de bicarbonato sódico en 100 ml H 2 O destilada </li></ol></li><li> Sumerja el portaobjetos en hematoxilina Gill II (0,4%) durante 5 minutos. </li><li> Lavar el exceso de manchas en constante grifo abierto H 2 O durante 3 min. </li><li> Sumerja la diapositiva 10 veces en ácido alcohol </li><li> Lave la diapositiva en el funcionamiento del grifo H2O durante 3 min. </li><li> Sumerja el portaobjetosen una solución de bicarbonato de sodio al 0,1% durante 30 a 60 segundos para encender el color morado a azul. </li><li> Lave la diapositiva en el funcionamiento del grifo H2O durante 3 min. </li><li> Enjuague el portaobjetos en etanol al 95%, con 10 inmersiones. </li><li> Sumerja el portaobjetos en tiempos Eosina-Y 2-3 durante 15 segundos totales. </li><li> Deshidratar el portaobjetos a través de etanol clasificado como sigue: sumergir el portaobjetos dos veces en etanol al 95%, dos veces en etanol al 100%, y dos veces en xileno (asegúrese de que el tejido parece claro que indica la deshidratación a fondo). </li><li> Montar la diapositiva con no acuoso dura medio de montaje y la cubierta antideslizante permanente. </li><li> Deje que el portaobjetos seco durante 30 minutos. </li><li> Diapositiva Scan con cualquier escáner de diapositivas entera e imprimir una imagen de gran tamaño, de alta resolución en 8 &quot;x 11&quot; de papel. </li><li> Áreas Esquema de interés (generalmente realizadas por un patólogo certificado por el consejo) con un marcador. Este marcado es la guía para el procedimiento de LCM. </li></ol> 3. toluidina azul tinción de diapositivas PEN-frame Nota: Jes que se hace el mismo día de la LCM. Utilice DEPCH 2 O el agua para todas las soluciones. Completar todos los pasos en la zona libre de RNasa. Todos los coplins deben ser limpiados con una solución de RNasa descontaminar. <ol><li> El día de LCM eliminar las diapositivas PEN-marco de -80 ° C congelador y de hidratos de diapositivas que sumergen suavemente en el 90% de EtOH durante 2 minutos y luego 75% EtOH durante 2 min. <ol><li> Preparar 0,5% de azul de toluidina por disolución en agua de calidad para biología molecular luego filtrar esterilizar a través de un filtro de 0,2 micras y almacenar a TA. Esta solución puede ser reutilizado para hasta 6 meses. Nota: Es muy recomendable para preparar la solución antes de que la filtración puede tomar unos pocos horas. </li></ol></li><li> Manchar el tejido de la próstata mediante inmersión suavemente diapositivas en 0,5% de azul de toluidina para 5 a 30 seg. Destain el tejido se desliza por lavado 2 veces en DEPCH 2 O durante 15 segundos seguido de 75% de EtOH durante 30 segundos a 3 min. Nota: las manchas y destain veces se pueden ajustar para asegurar una buena tinción. Próstata tiende a más de mancha. </li><li&gt; Transferencia de diapositivas PEN-marco para recipiente seco ARNasa libre de hielo. </li></ol> 4. láser captura micro-disección (LCM) <ol><li> Inmediatamente antes de la LCM, tobogán seco en un calentador de portaobjetos a 42 ° C durante 15 min. Seco sólo la diapositiva que se procesa y almacena el resto en hielo hasta que esté listo para ellos. </li><li> Encienda el LCM y el ordenador en este orden, el poder del microscopio, la clave de láser, el interruptor láser y luego la computadora. Inicie el software Láser microdisección. </li><li> Utilice el software para controlar el microscopio para cargar los toboganes y tubos de recolección. Haga clic en &quot;soporte de muestra de descarga&quot;, cargue la diapositiva con las secciones en el soporte de portaobjetos de microscopio con el tejido hacia abajo e inserte el soporte de diapositivas en la ranura correspondiente en el microscopio, a continuación, haga clic en &quot;Continuar&quot;. <ol><li> Haga clic en &quot;tubos de recogida de descarga&quot;, etiqueta y cargar 0,5 ml tubos en soporte de tubo y la inserta en la ranura correspondiente en el instrumento. Una veztapas están asegurados, añadir 35 l de tampón de lisis a las tapas de recolección y tratar de evitar burbujas. Luego haga clic en &quot;Aceptar&quot;. Nota: Si la evaporación es un problema, diluir el tampón de la siguiente manera: tampón de lisis 25 l con 10 l depcH2O. </li></ol></li><li> En el software, seleccione la posición en la diapositiva se ha colocado en el soporte de diapositivas. Seleccione la tapa de recogida para recoger la muestra de LCM. </li><li> Visualice y enfocar la diapositiva a través del ordenador a 10X. Utilice el software para calibrar el láser mediante la selección de &quot;láser&quot; y luego &quot;calibrar&quot;. Ajustar la potencia, apertura y la velocidad del láser, la selección de &quot;láser&quot; y luego &quot;control&quot; para permitir que el rayo láser para cortar la zona seleccionada por completo y de manera eficiente. Repita los ajustes hasta que el láser corta completamente a través del tejido. </li><li> Utilizando el margen de ganancia patólogo 8 &quot;x 11&quot; como guía, utilice la herramienta de &quot;llamar la forma de&quot; circunscribir las áreas deseadas de epitheLium en la vista y evitar recoger cualquier estroma. Nota: Las zonas Se pueden seleccionar varios dentro de un punto de vista, pero no se mueven a otra vista sin antes cortar haciendo clic en la herramienta &quot;cortar la forma&quot;. <ol><li> Si un área no quede completamente aislada por el láser utilizar la función &quot;mover y cortar&quot; para repasar de forma manual. Continuar mover el objetivo a nuevos puntos de vista y repetir el corte por láser hasta corredera está completamente disecado o la cantidad deseada de tejido que se ha recogido (típicamente 100 glándulas benignas producen de 150-500 ng de ARN). (véase el ejemplo en la figura 1A). </li></ol></li><li> Retire con cuidado los tubos de la titular de la recolección y cerrar las tapas, siendo consciente del tampón de lisis y tejidos en las tapas. Centrifugar brevemente durante 30 segundos para recoger el líquido y el tejido en la parte inferior del tubo. Incubar las muestras a 42 ° C durante 30 minutos y se almacena a -80 ° C hasta que esté listo para el aislamiento de ARN. </li></ol> 5. Aislamiento de ARN total con un FilKit basado en ter y Análisis de la Calidad <ol><li> Descongelar los tubos en hielo y llevar el volumen de la solución a 100 l. </li><li> Pre-humedecer el filtro de micro de un kit de aislamiento de ARN mediante la aplicación de 30 l Solución ofLysis al centro del filtro y déjelo en remojo whileperforming los dos pasos siguientes (al menos 5 min). </li><li> Añadir 3 l de solución de LCM Aditivo (proporcionado en el kit) al lisado y mezclar mediante agitación durante 5 s. Centrifugar durante 30 segundos para recoger el fluido en la parte inferior del tubo. </li><li> Centrifugar el filtro pre-humedecida por unos 30 segundos a máxima velocidad para eliminar el líquido. </li><li> Añadir 1,25 volúmenes (en este caso 129 l) de etanol al 100% a la mezcla de lisado, suavemente vórtice o una pipeta de arriba abajo. ** Este método se recuperará ambas especies de ARN grandes y pequeñas. </li><li> Cargar la muestra sobre el filtro micro-pre mojada, y se centrifuga a 10.000 xg durante 1 min para unir el ARN al filtro. A continuación, lavar micro filtro con 180 l de &quot;solución de lavado 1, R21; centrifugar a 10.000 xg durante 1 min. Nota: A partir de ahora todas las centrifugaciones se debe hacer a 13.000 xg, o la velocidad máxima. </li><li> Filtro de lavado dos veces con 180 l de &quot;soluciones de lavado 2 y 3&quot;, respectivamente según las indicaciones del protocolo del kit. Centrifugar durante 30 segundos, a continuación, se descarta el flujo a través, y girar el filtro durante 1 min para eliminar el líquido residual y para secar el filtro. </li><li> Transferencia del filtro a un nuevo tubo de recogida. </li><li> Caliente la solución de elución a -95 ° C (suministrado con el kit). </li><li> Aplicar 10 l de solución de elución precalentado al centro del filtro, cerrar la tapa y almacenar durante 5 min a RT. Centrifugar a continuación durante 1 min para eluir el ARN, y repetir este paso una vez para dar ~ 20 l de ARN. </li><li> Realizar el tratamiento DNasa I durante 15 minutos a 37 ° C. </li><li> Cuantificar el ARN mediante el espectrofotómetro con la absorbancia a 260 nm. La relación de la densidad óptica a longitudes de onda de 260 nm y 280 nm se utiliza para evaluar la pureza deel ARN (ver Tabla 1) 15. </li></ol> Análisis de Expresión Génica 6. Nota: La expresión de genes de especies de ARN largos (como los ARNm y lncRNAs) se muestra en el paso 6.1 y especies de ARN cortas (como microRNAs) se muestra en el paso 6.2. Diferentes kits están disponibles para RT-qPCR y la cantidad de ARN requerido varía según el kit (tan poco como 10 ng). Análisis de secuenciación de ARN se describe en 6.3. <ol><li> Transcribir inversa (RT) del RNA a cDNA mezclando 5 l de 20 ng / l de muestra de ARN con 5 l de la mezcla maestra RT (tampón RT, dNTPs, cebadores aleatorios, enzima RT, inhibidor de RNasa). Se incuba la reacción durante 25 min a 25 ° C, a continuación, 120 min a 37 ° C y 5 min a 85 ° C. </li><li> Se diluye la reacción RT 1:10 con RNasa libre de agua y el uso de 2,5 l por 10 l de reacción qPCR con cebadores o conjuntos de cebadores / sondas para los genes de interés. Nota: Para facilitar el análisis de ARN parcialmente degradado Es aconsejable el uso de cebadores for amplicones más cortos (menos de 100 bp) 16. (Tabla 2B) <ol><li> Para el análisis de la expresión de microARN ejecutar la reacción de RT mezclando 5 l de 2-10 ng / l muestra de ARN con 5 l de la mezcla maestra RT. Nota: cada tecnología de detección de microRNA disponible comercialmente basado en la PCR requiere el uso de cebadores de RT de propiedad y específicas. </li></ol></li><li> Alternativamente o además de RT-PCR, utilizar la secuenciación de próxima generación (RNA-Seq o pequeño-RNA-Seq) para cuantificar las diferentes especies de ARN. Típicamente, utilice 500 ng de ARN para este procedimiento (Tabla 3) y llevar a cabo el método como se indica por el instrumento fabricante y detección. </li></ol>

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The authors have nothing to disclose.

Perfiles de expresión génica de muestras humanas puede ser un reto, no sólo por la calidad o cantidad de tejido disponible, sino también por las diversas entidades histológicas presentes en una muestra de tejido dado. Esto es particularmente difícil en la próstata en el que los tejidos benignos son en gran parte los tejidos del estroma y áreas de cáncer están desprovistos de estroma. LCM facilita la separación física de estroma prostático y ARN epitelio para una firma más precisa de los dos tipos diferente...

La glándula prostática es un tejido heterogéneo compuesto de epitelio secretor dispuestos en los acinos glandulares rodeado por estroma fibromuscular compuesto principalmente de músculo liso 1. El compartimiento epitelial está formado por cinco tipos de células diferentes, pero organizados: células basales, células secretoras, células neuroendocrinas, células de amplificación de tránsito y las células madre 2. En el cáncer de próstata (CaP), que surge de las células epiteliales luminales, el crecimiento de la adenocarcinoma provoca una disminución progresiva evidente del compartimiento del estroma 3. Por estas razones, las muestras de tejido tendrán claras diferencias en la proporción de estroma y el tipo de células epiteliales basado en la medida del CaP. Estas diferencias pueden conducir a suposiciones sesgadas de los datos de expresión de genes adquiridos de tejidos enteros sin tener en cuenta microdisección del tipo celular deseado. Por lo tanto, para eliminar este sesgo es esencial para separar los tipos de células antes de la extracción de RNA yanálisis de expresión génica. Macrodissection o microdisección se pueden utilizar para separar físicamente las zonas epiteliales bien caracterizados desde el estroma circundante 4 -6. Macrodissection se realiza típicamente con una hoja de afeitar bajo un microscopio de disección y funciona bien para la separación de gran CaP nódulos de estroma, pero no es capaz de eliminar el epitelio benigno del estroma circundante (véase el ejemplo de la histología benigna de la próstata en la Figura 1). Microdisección con láser (LCM) es significativamente más mano de obra que macrodissection, pero puede diseccionar de forma muy precisa el epitelio benigno 4. Publicaciones recientes de nuestro laboratorio han demostrado que el ARN se puede extraer con éxito por LCM ya sea de (FFPE) biopsias incluidas en parafina fijado en formol o tejido congelado 4,7-9. Los principales desafíos en la extracción de RNA son LCM-1) para diseccionar con precisión las áreas deseadas del tejido, y 2) para preservar RIntegridad NA durante el proceso de 4,10 LCM y el aislamiento. ARN aislado de poblaciones de células puras puede ser utilizado para el análisis de la expresión génica mediante varios métodos que incluyen transcripción inversa-PCR cuantitativa (RT-qPCR) 7,8, microarray 11, y profundo -sequencing 12-14. El objetivo de este protocolo es aislar ARN total de epitelio prostático LCM de tejido congelado para análisis de aguas abajo de la expresión génica.

<table>
	<tbody>
		<tr>
			<td>RNase-AWAY&#160;</td>
			<td>MBP</td>
			<td>7005-11</td>
			<td></td>
		</tr>
		<tr>
			<td>PEN-membrane 4,0 mm slides&#160;</td>
			<td>Leica</td>
			<td>11600289</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides, Superfrost Plus</td>
			<td>FisherBrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon labs.</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC (diethyl pyrocarbonate)</td>
			<td>Sigma</td>
			<td>D-5758</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplins (Staining jar)</td>
			<td>IHCWORLD</td>
			<td>M900-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplins (Staining rack)</td>
			<td>IHCWORLD</td>
			<td>M905-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Aperio ScanScope</td>
			<td>Aperio(Leica)</td>
			<td>ScanScope&#174; CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Fluka</td>
			<td>89640-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Microdissection System</td>
			<td>Leica</td>
			<td>LMD7000</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL Thin-walled Tubes for LCM</td>
			<td>Thermo Scientific</td>
			<td>AB-0350</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAqueous&#174;-Micro Total RNA Isolation Kit</td>
			<td>Ambion</td>
			<td>AM1931</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Scientific</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 Fluorometer</td>
			<td>Life Technologies</td>
			<td>Q32866</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Applied Biosystems</td>
			<td>4368814</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>Universal cDNA Synthesis Kit II, 8-64 rxns</td>
			<td>Exiqon</td>
			<td>203301</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan microRNA RT kit</td>
			<td>Applied Biosystems</td>
			<td>4366597</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>Hematoxylin stain&#160;</td>
			<td>Ricca Chemical Company</td>
			<td>3536-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin-Y</td>
			<td>Richard Allan Scientific&#160;</td>
			<td>7111</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser-capture microdissection of human prostatic epithelium for rna analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

En un estudio previo se demostró el uso de LCM para recoger tejidos epiteliales y del estroma de comparar los perfiles de expresión por RT-qPCR de mRNA y microRNA de los tejidos de la próstata congelados y FFPE del mismo paciente 4. LCM es mucho tiempo, sobre todo si gran cantidad de ARN se percibirá para el análisis de secuenciación de próxima generación. Por lo tanto, es crucial para mantener el espacio de trabajo y las herramientas de RNasa libre. Se recomienda examinar los controles de calidad y c...

Watch this Scientific Journal Video about Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis at JoVE.com

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Laser-captura microdisección de Human Prostática epitelio de Análisis de ARN

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

laser-capture-microdissection-human-prostatic-epithelium-for-rna

Research

JoVE Journal

Medicine

13.3K vistas.  University of Illinois at Chicago. The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Video: Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

La disección de los elementos humanos Cuerpo Vítreo para el análisis proteómico

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

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 ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

La formación del epitelio prostático humano utilizando tejido recombinación de roedores Urogenital mesénquima seno y células madre humanas

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

Renal Cápsula xenotrasplante y subcutánea Pellet Implantación para la Evaluación de la próstata Carcinogénesis y la hiperplasia prostática benigna

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Estudiar la Diabetes a través de los ojos de un pez: Microdissection, visualización y análisis de la vasculatura de retina de pez cebra de adulto tg(fli:EGFP)

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the quality of life. BPH includes 2 major treatments: medication and surgery. Surgical treatment is often the most effective and final intervention. Holmium laser enucleation of the prostate (HoLEP), one of the most efficient surgical procedures for BPH, is conducted transurethrally. During surgery, the most important and most difficult part is to locate the surgical capsule of the prostate, which is consistent to the idea of anatomic surgery. Different skills may be needed in treating different parts of the prostate. HoLEP tends to be efficient and safe with good hemostatic properties, and able to treat bladder calculus, which may be the complications of BPH. The technique is particularly suitable for prostate of varying volumes and sizes. Indeed, HoLEP also presents certain disadvantages, such as a long learning curve and costly equipment. Regardless, this method may be the "new standard" and the excellent method for the surgical treatment of BPH.

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate HoLEP.

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

Tratamiento quirúrgico de la hiperplasia prostática benigna: Holmium láser enucleación de la próstata (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Cuantificación espacial de fármacos en lesiones de Tuberculosis pulmonar por Laser Capture Microdissection líquido cromatografía espectrometría de masas (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Nanopartículas de óxido de hierro recubierto de polietilamina como vehículo para la entrega de ARN interferente pequeño a los macrófagos In Vitro e In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Aislamiento de adipocitos viables y fracción vascular estromal a partir de tejido adiposo visceral humano adecuado para análisis de ARN y fenotipado de macrófagos

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Trasplante de epitelio pigmentario retiniano en un modelo de primate no humano para enfermedades degenerativas de la retina

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...