Este trabajo fue apoyado por subvenciones del Instituto Nacional de Salud-SR1 GM085130 y National Science Foundation MCB0929394. Agradecemos a la Dra. Marion Schmidt por su hospitalidad y por permitirnos filmar en su laboratorio.

1. Preparación de los reactivos Huella <ol><li> Preparar un tampón de reacción 10x con 100 mM cacodilato de sodio, 1 mM EDTA y 1 M KCl. Ajustar el pH a 7.4. Filtro de la memoria intermedia con un 0,2 M de acetato de dispositivo de filtro (Nalgene). Nota: no ARN pipeta directamente en el buffer 10x. </li><li> Prepare la mezcla de reacción de titulación para cada reacción, como se indica en la Tabla 1. El volumen de la mezcla de la valoración (1x buffer y Mg (II) a la concentración deseada) debe ser de 90 l, antes de añadir 10μl de ARN en tampón 1x. </li><li> Preparar un buffer de RNasa T1 digestión que contiene urea 6.63M, citrato de sodio 20 mM, 1 mM EDTA, 0,25 mg / l tRNA, 0,025% cyanol xileno, y 0,025% de azul de bromofenol. Este buffer se puede almacenar a 4 ° C durante un máximo de 6 meses. </li><li> La reacción de Fenton requiere recién preparada las soluciones acuosas de 250 mM Fe (NH 4) 2 (SO 4) 2, 250 mM EDTA y 500 mM de sodio L-ascorbato. Las concentraciones en el capítulo 10.5 son las más adecuadas para los experimentos de equilibrio en un tampón de cacodilato de sodio, tales como que no se eliminan los radicales y diversas concentraciones de iones divalentes. </li><li> Los reactivos para la reacción de la huella peroxidativo (1.5.1) y oxidativo (1.5.2). <ol><li> La peroxidación reacción de Fenton requiere recién preparada las soluciones acuosas de 100 mM Fe-EDTA, y 50 mM de sodio L-ascorbato y el 1,5% H 2 O 2. La solución de Fe-EDTA se prepara mezclando Fe (NH 4) 2 (SO 4) 2 con EDTA a tener 1,1 veces el exceso de EDTA sobre el hierro. </li><li> Justo antes de iniciar la reacción de escisión oxidativa (3.4.2), crear la mezcla de reacción de Fenton mediante la combinación de 2 l de Fe (NH 4) 2 (SO 4) 2 (250 mm), 2,2 l de EDTA (250 mM), 62,5 l L-ascorbato sódico (500 mM), 22,5 l buffer de reacción 10x y 135,5 l de H 2 O para llegar a la concentración final en la mezcla de reacción de huellas (3.4.2) de 0.10 mM, 0,11 y 6,61 mM, respectivamente. </li></ol></li></ol> 2. ARN preparación para el experimento Huella <ol><li> ARN puede ser producido por la norma en la transcripción in vitro de seis plantillas de ADN o, si menor de 50 nucleótidos, pueden comprar (por ejemplo, en Integrated DNA Technologies, www.idtdna.com). Siga los procedimientos recomendados para la limpieza de laboratorio para mantener la integridad de las muestras de ARN durante los experimentos huella 7. </li><li> 8 de desfosforilación in vitro ARN transcrito es necesaria antes de etiquetado P 32 del extremo 5 &#39;del ARN. 10 pmol de RNA son finales etiquetados por kinasing. 9 La solución debe ser despejado con éxito después de la inactivación de la fosfatasa. </li><li> Purificar el ARN marcado radiactivamente mediante electroforesis en gel de desnaturalización. El ARN se recupera por dos posteriores extracciones de gel con 0,3 M de acetato de sodio (pH 5,5). ARN se precipita mediante la mezcla de 0,5 ml de solución acuosa con 1 ml de etanol y la posteriorincubación durante 1 minuto en hielo seco. Giro de la muestra a 4 º C, 12500 rpm durante 0,5 horas. Descartar el sobrenadante y lavar el ARN precipitado con etanol al 70%. Aspirar el sobrenadante después de centrifugación adicional y seco en pelets en el vacío. </li><li> Disolver los 32 P-etiquetados muestras de ARN en 330 ul de 1x Buffer.Pipet 30 l de la solución de ARN en un tubo de reacción, el precipitado con etanol y el ARN en seco en el vacío, mantenga este tubo para la generación de la escala de referencia (paso 3.5). Desnaturalizar la solución tampón de ARN por calentamiento a 95 ° C durante 2 min. Fresco de las muestras a temperatura ambiente durante 15 min y dar una vuelta rápida para reducir la condensación en las tapas de los tubos de nuevo el producto. </li></ol> 3. El experimento de Huella <ol><li> El peine de electroforesis determina el número máximo de puntos de datos por gel analítica. Nosotros usamos un peine y 30. La escalera de referencia ocupa dos pozos y el control de uncleaved. Prepare 27 tubos de reacción. El final de Mg (II) Concentradoscomplementos deben ser espaciados uniformemente en una escala logarítmica que abarca varios órdenes de magnitud cerca del punto medio de valoración. </li><li> Por otra parte, se incuba el ARN y las soluciones de Mg (II) a 50 ° C durante 5 min. Mezclar 10 l de solución de ARN con 90 l de la cantidad correspondiente de Mg (II) en 1x buffer para alcanzar un volumen final de 100 microlitros. Incubar las soluciones durante 30 minutos a 50 ° C. </li><li> Deje que las soluciones de equilibrio a 25 ° C durante 1 hora, mientras que plegado tiene lugar. </li><li> Peroxidativo (3.4.1) o oxidativo (3.4.2) la reacción hidroxilo huella radical. <ol><li> Iniciar la reacción de peroxidación mediante la colocación de las gotas de 2 l de Fe-EDTA (100 mM), 2 l de sodio L-ascorbato (50 mM) y 2 l H 2 O 2 (1,5%), separadas unas de otras en el interior de la parte superior de la reacción tubo que contiene la solución de ARN e iniciar la reacción de la huella de una mezcla vigorosa. Detener la reacción después de 15 segundos mediante la adición de 300 l de etanol absoluto frío. A su vez los tubos de 3.5 veces. Precipitado, lavar ysecar el pellet como en el paso 2.4. </li><li> Inicie el footprintingreaction oxidativo de radicales hidroxilo mediante la adición de 5 l de la mezcla de reacción recién preparada Fenton (1,6). Incubar durante 30 min a 25 ° C. Añadir 300 l de etanol absoluto frío para detener la reacción y mezclar girando el tubo 3-5 veces. Precipitado, lavar y secar el pellet, como se indica en el paso 2.4. </li></ol></li><li> Generar una RNasa T1 muestra de referencia digerir acuerdo con los procedimientos estándar de 10 con la solución preparada en el paso 1.3. </li><li> Disolver pellets de ARN en 8 l de gel de carga de tintes II (Ambion) y el ARN se vuelve a suspender confirmar mediante el contador Geiger. </li><li> Prepare una desnaturalización, gel de poliacrilamida al 8% de secuenciación de acuerdo con protocolos estándar. 11 muestras de carga, incluyendo dos referencias y un control. Separar fragmentos de ARN a 60 W - 75 W durante 2,5 horas. Exponer gel seco a una pantalla de fósforo de almacenamiento durante la noche. Exploración pantalla de fósforo con un sistema de imagen para autorradiografía sin película, por ejemplo,. Tormenta 865 (GE Healthcare) o tifón (GE Healthcare). Transferencia de archivos gel imagen en tu ordenador. </li></ol> 4. Análisis de Datos <ol><li> La reactividad del radical hidroxilo de cada nucleótido se determina mediante la cuantificación de la intensidad de cada banda en el gel de análisis. Descargue, instale y abra SAFA, un fácil utilizar el software de código abierto para montar una sola banda y cuantificación. 12, 13 Siga las instrucciones del manual del usuario. En pocas palabras, la carga de la secuencia de ARN como. Txt seguido de la imagen como un archivo de gel. Gel. Ajustar la intensidad de la banda, definir los carriles, elija un carril de anclaje, y realizar una alineación gel. Asignación de bandas de nucleótidos ocurre en referencia a la escalera de RNasa T1 digestión. Cuantificar las integrales de banda y usar la función de normalización / colorplot para normalizar y asignar los residuos potencialmente invariante. Guardar el resultado como un archivo. Txt. </li><li> SAFA salidas de una hoja de cálculo que contiene las columnas que representan los carriles en el gel y las filas que representan entegrated banda de densidad de las bandas individuales correspondientes a los fragmentos de ARN. En primer lugar, identificar los sitios de protección que muestran un cambio notable en la accesibilidad de disolvente mediante la comparación del perfil de la protección derivada de la no Mg (II) de la muestra con el perfil de el punto final de Mg (II) de concentración (50 mM en nuestro ejemplo). Cuanto más bajo sea el valor, más protegido es el nucleótido está en contra del ataque de los radicales hidroxilo y viceversa. </li><li> Crear isotermas plegado de las integrales de banda normalizado de las personas o grupos de nucleótidos frente a Mg (II) de concentración. Las isotermas son individualmente a escala de la saturación fraccional por fi = L + (U - L) • <img src="files/ftp_upload/3244/3244y.jpg" alt="Y" /> donde f denota la densidad integrada de la banda (s) que se analiza, L y U representan los límites inferior y superior de la transición y <img src="/files/ftp_upload/3244/3244y.jpg" alt="Y" /> es la saturación fraccional. 14 Los datos se ajustan utilizando una l no linealEste programa de análisis de cuadrados (Usamos ya sea de origen (OriginLab) o GraphPad (GraphPad Software, Inc.).) a la ecuación de Hill (1), <img alt="La ecuación 1" src="/files/ftp_upload/3244/3244eq1.jpg" /> donde K d es el equilibrio constante de disociación, [M] corresponde a la concentración de la variable que interviene en la reacción de plegamiento, Mg (II) en este ejemplo, y H n es el coeficiente de Hill. Este procedimiento de escalas de la transición a la saturación fraccional, determina su punto medio y proporciona una prueba fenomenológica de si la transición es sigmoidal. Isotermas derivadas de geles de secuenciación (Figura 3), que fueron analizados por SAFA (Figura 3B) y el ajuste como se describe anteriormente se muestran en la Figura 3C. Las isotermas de saturación fraccional se muestra en la Figura 3 se generaron por la escala en una hoja de cálculo de los valores generados por la SAFA contra el mejor ajuste de los límites superior e inferior (U y L) con la fracción de la ecuación = valor / (U - L) - L / (U - L). </li></ol> 5. Los resultados representativos: La Figura 3 muestra los resultados representativos de P4-P6 ARN • Experimentos huella OH. La imagen de gel (izquierda) indica que una división de fondo) es mínimo (carril derecho), b) la asignación de un solo nucleótido es posible gracias a las bandas bien definidas en el carril de la T1 (RNasa T1 se unirá en cada G), y c) el radical hidroxilo la fragmentación inducida por el ARN está muy por encima del fondo. La transición de bajo a alto Mg (II) está afiliada con la disminución de la densidad de la banda integrada de individuos y grupos de bandas que indican la formación de la estructura del ARN terciario. Individuales o grupos de nucleótidos contiguos cuya división cambios de forma concomitante se conoce como &quot;protección&quot;. Las protecciones • OH características de la Mg (II) mediada plegado de la ARN P4-P6 corresponden muy de cerca a las regiones inaccesibles solvente de la molécula de observar en su estructura cristalina. 15 Doblado las moléculas de ARN c<html> uno tiene extensiones muy diferentes (es decir, qué tan cerca a un segundo plano la declinación banda densidades) de protección contra el contacto superior que no se han relacionado con el fenómeno de clara estructurales o dinámicas. Por lo tanto, algunas moléculas de ARN se muestran bien definidas las transiciones estructurales, como se muestra en la Fig. 3, y otros no. Intensidades de las bandas se cuantificó y se normalizó por la SAFA 12 análisis (Figura 3B). La salida es un &quot;térmico&quot; complot visualizar el grado relativo de la protección contra • OH. La transición de colores describe el cambio de la accesibilidad en Mg (II) Además. Blanco a rojo o azul muestra nucleótidos más accesible o más protegidas, respectivamente. Cada grado de sombreado está afiliado a un valor numérico que se pueden representar como una curva de protección (Figura 3C) y se analizaron mediante un modelo de unión, como la ecuación de Hill (1). La constante de equilibrio de disociación afiliado con la protección del 153-155 es aproximadamente el doble que el valor correspondiente de protección 163-164. CONTENIDO &quot;&gt; <img alt="Figura 1" src="/files/ftp_upload/3244/3244fig1.jpg" /> Figura 1. Producción de radicales hidroxilo y P4-P6 plegamiento del ARN. A) Fe (II) cataliza la generación de radicales hidroxilo a partir de peróxido de hidrógeno. Ascorbato reduce Fe (III) de vuelta a hierro ferroso. B) En ausencia de Mg (II), sin estructura terciaria P4-P6 se ​​forma, lo que los radicales hidroxilo para penetrar y romper todas las posiciones de columna vertebral accesible. Además de la Mg (II) inicia plegamiento de P4-P6, permitiendo sólo la columna vertebral accesible solvente para ser cortado por los radicales hidroxilo. <img alt="Figura 2" src="/files/ftp_upload/3244/3244fig2.jpg" /> Figura 2. Esquema de un experimento de la huella de radicales hidroxilo. A Dephophorylation) y 32 P 5 &#39;final de etiquetado de ARN. B) La purificación de 32 P-RNA marcado en un gel de poliacrilamida. C) La escisión de la banda de ARN, tras la extracción de ARN, y precipitación con etanol. D) Prefolding yplegamiento del ARN. E) La adición de recién preparada mezcla de reacción de Fenton para generar radicales hidroxilo. F) ARN separación fragmento de desnaturalización electroforesis en gel de poliacrilamida. G) La cuantificación de fragmentos de ARN por el software de SAFA. <img alt="Figura 3" src="/files/ftp_upload/3244/3244fig3.jpg" /> Figura 3. Análisis de fragmentos de ARN después de la reacción peroxidativa huella Fenton. (A) El ARN fue expuesto a los radicales hidroxilo y los productos de degradación se resolvieron mediante electroforesis en gel desnaturalizante de poliacrilamida (PAGE). La imagen muestra los productos de peroxidación de separación con la creciente concentración de Mg (II). Carriles de referencia y control están etiquetados. Este archivo de imagen es la entrada para el programa SAFA que cuantifica el volumen de cada banda. (B) El &#39;térmico&#39; trama generada por la SAFA. (C) Una hoja de cálculo de los valores relacionados con la densidad de la banda integrada a la salida número de nucleótidos de SAFA es analizada por un modelo de unión, como el Cerro equatien (1). Regiones de protección fueron escogidos de acuerdo con el aumento de la densidad de banda integral en función de Mg (II) de concentración. El K d es el Mg (II) concentración en la que se dobla la mitad de la ARN en el sitio se está supervisando. El coeficiente de Hill, n H, es una medida de la pendiente de la curva que da información sobre la cooperatividad de unión y proporciona una estimación más baja para el número de iones de magnesio que participan en el plegamiento de este sitio en particular. <img alt="Tabla 1" src="/files/ftp_upload/3244/3244table1.jpg" /> Tabla 1. Representante para la generación de volúmenes de muestras de ARN que contiene diferentes Mg (II) las concentraciones.

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No hay conflictos de interés declarado.

La huella radical hidroxilo es una herramienta valiosa para evaluar el área de superficie accesible disolvente de los ácidos nucleicos. Formación cualitativa y cuantitativa de la estructura terciaria 14 se puede seguir en función de parámetros como el tipo de iones y la concentración, pH, temperatura, proteínas de unión o plegamiento factores de co-. La atractiva combinación de un protocolo sencillo y de bajo costo y la accesibilidad resulta solvente y la información de plegado en un nivel de nucle?...

<table border="1">
	<tbody>
		<tr>
			<th>Name</th>
			<th>Company</th>
			<th>Cat#</th>
		</tr>
		<tr>
			<td>Sodium Cacodylate
			(Caution! Toxic)	 	
			</td>
			<td>Sigma</td>
			<td>C4945-25g</td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Ambion</td>
			<td>AM9260G</td>
		</tr>
		<tr>
			<td>DEPC treated water</td>
			<td>Ambion</td>
			<td>AM9915G</td>
		</tr>
		<tr>
			<td>Sodium Acetate (3 M)</td>
			<td>Ambion</td>
			<td>AM9740</td>
		</tr>
		<tr>
			<td>MgCl2 (1 M)</td>
			<td>Ambion</td>
			<td>AM9530G</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Ambion</td>
			<td>AM9902</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Sigma</td>
			<td>W302600</td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>Sigma</td>
			<td>R-7876</td>
		</tr>
		<tr>
			<td>Sodium-L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25g</td>
		</tr>
		<tr>
			<td>Fe(NH4)2(SO4)2 . 6 H2O</td>
			<td>Sigma</td>
			<td>F1543-500g</td>
		</tr>
		<tr>
			<td>RNase T1</td>
			<td>Fermentas</td>
			<td>EN0541</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (30%)</td>
			<td>Sigma</td>
			<td>349887</td>
		</tr>
	</tbody>
</table>

monitoring equilibrium changes in rna structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting

Las moléculas de ARN desempeñan un papel esencial en la biología. Además de transmitir la información genética, el ARN puede plegarse en estructuras terciarias única que cumple un papel biológico específico, como regulador, carpeta o un catalizador. Información acerca de la formación terciaria de contacto es esencial para entender la función de moléculas de ARN. Radicales hidroxilo (OH •) son las sondas único de la estructura de los ácidos nucleicos, debido a su alta reactividad y tamaño pequeño. 1 Cuando se utiliza como una sonda de huellas, los radicales hidroxilo mapa de la superficie accesible al soluto de la columna vertebral fosfodiéster del ADN y el ARN 1 2 con tan fina como la resolución de un solo nucleótido. La huella radical hidroxilo se puede utilizar para identificar los nucleótidos dentro de una superficie de contacto intermolecular, por ejemplo, en el ADN en proteínas y complejos de un ARN-proteína. Equilibrio 3 y 4 cinética transiciones se puede determinar mediante la realización de la huella de radicales hidroxilo en función de un solutien la variable o la hora, respectivamente. Una característica clave de la huella es que la exposición limitada a la sonda (por ejemplo, &quot;un solo ataque cinética&quot;) se traduce en la toma de muestras uniformes de cada nucleótido del polímero. 5 En este artículo, video, utilizar el dominio P4-P6 de la ribozima de Tetrahymena para ilustrar la preparación de ARN de la muestra y la determinación de Mg (II) mediada por las isotermas de plegado. Se describe la utilización del conocido protocolo de hidroxilo huella radical que requiere de H 2 O 2 (a esto lo llamamos el &quot;peroxidación&quot; protocolo) y un valor, pero no se conoce ampliamente, alternativa que utiliza naturalmente, O 2 disuelto (llamamos a esto &quot;el oxidativo &quot;protocolo). Una visión general de la reducción de datos, la transformación y los procedimientos de análisis se presenta.

Watch this Scientific Journal Video about Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting at JoVE.com

Monitoring Equilibrium Changes in RNA Structure by &#039;Peroxidative&#039; and &#039;Oxidative&#039; Hydroxyl Radical Footprinting

Este protocolo describe la forma de cuantificar el Mg (II)-dependiente de la formación de la estructura del ARN superior mediante dos métodos de huella radical hidroxilo.

Seguimiento de los cambios de equilibrio en la estructura del ARN por &quot;peroxidación&quot; y &quot;oxidativo&quot; Huella de radicales hidroxilo

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

monitoring-equilibrium-changes-rna-structure-peroxidative-oxidative

Research

JoVE Journal

Biology

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

14.2K vistas.  Hunter College. Este protocolo describe la forma de cuantificar el Mg (II)-dependiente de la formación de la estructura del ARN superior mediante dos métodos de huella radical hidroxilo.

Video: Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Métodos de una sola molécula para vigilar los cambios en las propiedades elásticas bicapa

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Investigación

Biología

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Seguimiento de los cambios dinámicos en niveles de calcio mitocondrial de la apoptosis mediante un sensor de calcio genéticamente codificados

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

En la transcripción in vitro y la nivelación de mRNA de luciferasa Gaussia Seguido por transfección de células HeLa

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A largo plazo Prueba de toxicidad letal con el Crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Aislamiento de la cromatina mediante la purificación de ARN (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

La detección de ARN viral por fluorescencia<em&gt; In situ</em&gt; La hibridación (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

ARN de predicción de estructura secundaria Utilizando SHAPE de alto rendimiento

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Reconstitución funcional y la actividad del canal Las mediciones de Purificada Wildtype y mutante CFTR Proteína

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

Seguimiento de cambios inducidos por fármacos en los receptores post-internalización Trata de Análisis Colocalizational

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Análisis de glucocorticoides fecales: Monitoreo suprarrenal no invasiva en los équidos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...