Nos gustaría agradecer a las siguientes personas, sin cuya ayuda la colección de tejidos de pino, no habría sido posible: el Dr. Joe Nairn, Bryman Matt, Michael Burdeos, Bagal Ujwal, Jin Huizhe y Bouffier Amanda.

Parte 1: Cosecha de árboles Después de que el árbol se selecciona y se cortan, es importante trabajar con cuidado y lo más rápidamente posible. Como cada tipo de tejido diferentes se cosecha, se deben colocar inmediatamente en sus propios barcos de nitrógeno líquido para evitar la contaminación cruzada de muestras. Este protocolo de aislamiento de ARN es escalable. <ol><li> Cortar los pernos en longitudes de aproximadamente 0,5-0,75 m y con una puntuación de motosierra de la corteza en dos o tres lugares, siguiendo el eje longitudinal del tornillo. </li><li> Utilizando un cincel de madera, el trabajo del borde de la región anotaron hasta la corteza comienza a separarse de la madera y seguir trabajando el cincel al tiempo que tira de nuevo en la sección de la corteza hasta que se separe por completo de la madre. </li><li> Xilema secundario se cosecha con un borde recto, de un solo lado de afeitar raspando a lo largo del eje longitudinal del tornillo que ha sido despojado de la corteza. Use guantes de látex para minimizar la contaminación de los tejidos. </li><li> Floema, que se encuentra en el lado interno de las secciones de la corteza, es fácilmente capturado por manual de pelar. </li><li> Madera de reacción se obtiene de la parte inferior de las extremidades después de marcarlos con pintura en aerosol para indicar su orientación original con respecto al suelo. </li><li> Las extremidades son cortadas en los tornillos de 2.1 m, y anotó en los lados opuestos a lo largo del eje longitudinal de acuerdo con la ubicación aproximada de los dos tipos de madera de reacción: madera opuesta (en la parte superior de la extremidad) y madera de compresión (en la parte inferior lado de la extremidad). La corteza se separa de la extremidad como se describió anteriormente, excepto que sólo la corteza que cubre cada tipo de madera de reacción se elimina de una vez. Xilema secundario o floema se recoge como se describe arriba y se colocan en nitrógeno líquido. </li><li> Otras muestras, tales como agujas, puntas de los brotes y los conos de los jóvenes, pueden ser cosechadas a mano o con tijeras de podar cuando sea necesario. </li><li> Las muestras recogidas se congelan en nitrógeno líquido debe ser colocado en bolsas o recipientes adecuados marcados y envasados ​​en hielo seco para que el traslado al laboratorio. </li></ol> Parte 2: Procesamiento de Freezer Mill de las muestras <ol><li> Llenar el depósito de molino Spex 6850 congelador de nitrógeno líquido y coloque la barra de impacto en uno de los viales de molienda y rellenar con nitrógeno líquido para pre-chill. Verter el nitrógeno líquido y añadir la muestra al vial. </li><li> Asegúrese de que el exceso de líquido de nitrógeno no permanece en la cámara de molienda y que el exceso de nitrógeno gas es ventilado completamente antes de que la tapa está sentado para sellar la cámara. </li><li> Introduzca el vial de molienda en el soporte y cerrar. </li><li> Molino de muestras de madera de dos ciclos de 1 minuto / ciclo en un ajuste de velocidad de 14. </li><li> Use una espátula de nitrógeno líquido refrigerado para extraer la muestra molida (harina de madera) en un vaso que ha sido pre-enfriado y contiene una pequeña cantidad de nitrógeno líquido. </li><li> Vierta el líquido de nitrógeno / muestra mezcla en un recipiente de almacenamiento y el lugar a -80 ° C hasta que se necesite. </li></ol> Parte 3: el aislamiento de ARN, el día 1 <ol><li> Coloque 20 ml de tampón de aislamiento de ARN en un tubo de 50 ml tapado Falcon y añadir 400 l β-mercaptoetanol, vortex brevemente, y luego el calor en un baño de agua a 60 º C. </li><li> Añadir 4 g de tejido congelado para cada tubo. Tapa y agitar a mano seguido por agitación durante 10 segundos. </li><li> Añadir un volumen igual de cloroformo en el tubo e invierta para mezclar suavemente. Coloque el tubo en una rueda giratoria y permitir que de extremo a extremo la mezcla durante 5 minutos. </li><li> Decantar la mezcla en un tubo de 50 ml de Oak Ridge y centrifugar a 12.000 (17.200 xg) rpm en un SS34 rotor de ángulo fijo durante 5 minutos. </li><li> Pasar la fase acuosa a un tubo de Oak Ridge fresco y añadir un medio volumen de cloroformo. </li><li> Vortex durante 1 minuto y seguir mezclando en una rueda giratoria durante 5 minutos. Centrifugar a 12.000 rpm (17.200 xg) en un rotor de ángulo fijo SS34 durante 5 minutos. </li><li> Pasar la fase acuosa a un tubo nuevo de Oak Ridge y añadir un medio volumen de cloroformo. Vortex durante 1 minuto. Centrifugar a 12.000 rpm (17.200 xg) en un rotor de ángulo fijo SS34 durante 10 minutos. </li><li> Pasar la fase acuosa a un tubo de 50 ml de polipropileno de alta velocidad. Centrifugar a 10.000 rpm (11.900 xg) en un rotor de ángulo fijo SS34 durante 20 minutos. </li><li> Decantar el sobrenadante en tubos Falcon de 50 ml y añadir un cuarto de volumen de 10 M a la solución de LiCl sobrenadante. Agitar brevemente y refrigerarlas a 4 ° C para la precipitación de la noche. </li></ol> Parte 4: el aislamiento de ARN, Día 2 <ol><li> Calor buffer SSTE en un baño de 65 ° C el agua antes de comenzar. </li><li> Vierta la muestra de LiCl precipita en un tubo de polipropileno y el ARN precipitado por centrifugación a 11.000 rpm en un rotor de ángulo fijo SS34 (14.450 xg) durante 30 minutos a 4 ° C. </li><li> Después de la centrifugación,Retirar y desechar el líquido, e invertir los tubos en Kimwipes durante aproximadamente 1 minuto. Pipeta de cualquier resto de sobrenadante. </li><li> Resuspender cada pellet en 800 l de SSTE calienta por medio de una pipeta para mezclar bien las muestras. Transferir la muestra se resuspendieron a 2 ml tubos de microcentrífuga Ambion RNasa libre. </li><li> Muestras de calor a 65 ° C durante 2 minutos, seguido por agitación vigorosa para asegurar la completa resuspensión. </li><li> Añadir un volumen igual de fenol-cloroformo, pH 8, a cada tubo de muestra. Vortex cada muestra durante 30 segundos y luego girar en una microcentrífuga a 14.000 rpm durante 3 minutos </li><li> Pasar la fase acuosa a un nuevo tubo de microcentrífuga de 2 ml Ambion RNasa libre, y añadir un volumen igual de cloroformo. Vortex cada muestra durante 30 segundos y girar en la microcentrífuga a 14.000 rpm durante 3 minutos. </li><li> Transferencia de fase acuosa a tubos de gel de enganche de fase, que han sido previamente preparados por microfuging a 11.000 rpm durante 2 minutos. Añadir la mitad de volumen de cloroformo y suavemente la pipeta para mezclar. </li><li> Centrifugar a 11.000 rpm durante 5 minutos y la transferencia de la fase acuosa a un nuevo tubo 2 ml de microfuga Ambion RNasa libre. </li></ol> Parte 5: ARN de precipitación y lavado de etanol <ol><li> Añadir 50 l de acetato de sodio 3,0 M, pH 4,8, y 1 ml de etanol al 100% a la fase acuosa. Vortex y precipitado en -80 ° C durante 30 minutos o toda la noche a -20 ° C. </li><li> Muestras de microcentrífuga a 14.000 rpm durante 30 minutos a 4 ° C, y cuidado aspirar el sobrenadante. No moleste a la bolita. </li><li> Lavar los gránulos de ARN con 1 ml -20 ° C 70% (v / v) de etanol. Microfuga a 14.000 rpm durante 1 minuto y aspirar el etanol. </li><li> Repita el paso 5.3. </li><li> Destape los tubos y los pellets de aire seco en el banquillo durante 3-5 minutos. </li></ol> Parte 6: resuspensión final y cuantificación <ol><li> Resuspender cada pellet en 100 l agua tratada con DEPC. Mezclar por agitación e incubar los tubos a 65 ° C durante 2 minutos antes de colocar en el hielo. Repita si es necesario para asegurar la resuspensión de la muestra. </li><li> Piscina como muestras si así lo desea y hacer 50 l de una dilución de 1:10 en 10 mM Tris, pH 7,5, para la cuantificación espectrofotométrica. Razón de absorbancia de 260/280 y 260/230 son típicamente mayores de 2.1 y 2.2, respectivamente. El aislamiento de ARN rendimientos oscilan entre 60-120μg / g de tejido congelado. </li><li> Analizar las muestras mediante la ejecución de 1,5-2,5 mg de ARN en un 1% en gel de agarosa en buffer TAE. Para más muestras de crítica, analizar el ARN utilizando un Agilent 2100 Bioanalyzer según las instrucciones del fabricante. </li></ol> Los resultados representativos: Los rendimientos de aislamiento de ARN de coníferas diferentes tejidos rango 60 a 120 mg / g de tejido congelado, con muestras de madera suelen ceder en torno al 70-80g / g de tejido congelado. Ratios de diagnóstico de 260/280 y 260/230 son por lo general mayor que 2.1, y son buenos indicadores de que la muestra de ARN está libre de cualquier contaminación o fenólicos importante de hidratos de carbono, respectivamente. Las muestras analizadas por electroforesis en gel de agarosa seguida de tinción EtBr debe mostrar tres bandas distintas de 25S, 18S y 5S ARN (Figura 1). La determinación de la estequiometría de la 28S a 18S en geles de agarosa es algo subjetivo, y factores tales como condiciones de funcionamiento para el gel, las manchas, y la carga de probar todos pueden tener un efecto. En general, el 28S: 18S relación se parecen ser&gt; 1,5. Sin embargo, algunas muestras de coníferas tienen menores proporciones. Por ejemplo, tras el análisis de Agilent 2100 hemos visto varios casos de muestras de coníferas de diferentes tipos de tejidos, donde la estequiometría está más cerca de 1,2:1. Por lo tanto, una aparente baja 28S: 18S estequiometría por electroforesis en gel de agarosa no indica necesariamente una muestra de mala calidad. Hoja, disparar, y las muestras de cono a menudo tienen más distintas bandas presentes debido a cloroplástico ARN ribosomal. EtBr manchado de materiales que no emigran de la muestra bien o bandas de alta masa molecular (&gt; 10.8 kb) por lo general indican la contaminación de ADN genómico (Figura 2). Frotis bajo peso molecular en las proximidades o debajo de la banda 5S y tinción reducida banda ribosomal 18S o un aparente&gt; estequiometría 28S es un buen indicador de que se ha producido la degradación del ARN (Figura 3). En el análisis electroferograma Agilent 2100, la línea de base debe ser baja y relativamente lisa, con picos principales correspondientes a los ARN 18S y 28S ribosomal 28S con: 18S ratios que van en cualquier lugar desde 1,2 hasta 2,0. El número de Agilent integridad del ARN (RIN) de valor puede ser un mejor predictor de la calidad del ARN y debe ser por lo menos 7 o superior para el ARN que se va a utilizar para la preparación de los objetivos de microarrays. La Figura 4 muestra una típica Agilent 2100 electroferograma para el ARN del xilema con un 28S: 18S proporción igual a 1,4 y un valor de RIN de 8,6 mientras que la Figura 5 se muestra un xilema pobres ARN de preparación con una base muy alta y la degradación notable como lo revela el 28S: 18S raigual a 0,65 y un valor de RIN de 3,4 tio. <img src="/files/ftp_upload/1751/1751fig1.jpg" alt="Figura 1" /> Figura 1. Agarosa análisis en gel de ARN de pino de alta calidad. Calle 1, ORC 1 kb estándar, Carril 2 muestra un típico aislamiento de ARN total de pino tea xilema secundario con características ribosomal 28S, 18S, 5S y las bandas y la aparente 28S: relación de 18S&gt; 1. <img src="/files/ftp_upload/1751/1751fig2.jpg" alt="Figura 2" /> Figura 2. Análisis en gel de agarosa de pino muestra de ARN contaminadas con ADN genómico. Calle 1, ORC 1 kb estándar, Calle 2, xilema muestra de ARN que muestra la contaminación de ADN genómico. <img src="/files/ftp_upload/1751/1751fig3.jpg" alt="Figura 3" /> Figura 3. Agarosa gel de análisis de la muestra de pino que muestra la degradación del RNA y de la contaminación con ADN genómico. Calle 1, ORC 1 kb estándar, Lane2, xilema muestra de ARN que muestra la contaminación de ADN genómico y severa degradación del ARN. <img src="/files/ftp_upload/1751/1751fig4.jpg" alt="Figura 4" /> Figura 4. Agilent 2100 electroferograma de ARN total xilema aislado utilizando el método descrito en el xilema pino tea. 28S: 18S relación de igual a 1,4, el valor de RIN equivale a 8,6 <img src="/files/ftp_upload/1751/1751fig5.jpg" alt="Figura 5" /> Figura 5. Agilent 2100 electroferograma de ARN total de la punta apical que muestra una severa degradación y la contaminación genómica. 28S: 18S relación de igual a 0,65, el valor de RIN equivale a 3,4

<ol>
	<li>Chang, S., Puryear, J., Cairney, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+method+for+extracting+RNA+from+pine+trees.">A simple and efficient method for extracting RNA from pine trees.</a> Plant Molecular Biology Reporter. 11 (2), 113-116 (1993).</li><li>Lorenz, W. W., Yu, Y. S., Sim&#369;es, M., Dean, J. F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+the+Loblolly+Pine+PtGen2+cDNA+Microarray.">Processing the Loblolly Pine PtGen2 cDNA Microarray.</a> Journal of Visualized Experiments. 25, (2009).</li></ol>

La obtención de ARN de alta calidad a partir de especies de coníferas puede ser una tarea difícil debido a los altos niveles de compuestos fenólicos y polisacáridos se encuentran en los tejidos leñosos. Comenzando con el protocolo desarrollado por Chang et al. (1), hemos encontrado que la extracción de más riguroso y limpieza de los pasos conducen al aislamiento constante de muy alta calidad total de ARN de varios tejidos leñosos no maderables en la muestra de una gran variedad de especies de coníferas. Este v...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>RNA Isolation Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2% CTAB (hexadecyltrimethylammonium bromide), 2% PVP (polyvinyl pyrrolidinone; Mw 30-40,000), 100mM Tris-HCl (pH8.0), 25mM EDTA, 2.0M NaCl, 0.5g/L Spermidine</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>C298-4</td>
<td>&nbsp;</td>
<tr>
<td>Phenol, Ultrapure</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15509-037</td>
<td>&nbsp;</td>
<tr>
<td>10M LiCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
</tr>
<tr>
<td>SSTE Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1M NaCl, 0.5% SDS, 10mM Tris-HCl (pH8.0), 1mM EDTA (pH8.0)</td>
</tr>
<tr>
<td>Sodium acetate (NaOAc) 3M pH4.8</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
</tr>
<tr>
<td>Phenol-chloroform (pH8.0)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>120mL phenol, 160mL chloroform titrated with multiple changes of 0.5M Tris-Cl pH 8.0</td>
</tr>
<tr>
<td>Oak Ridge High Speed Teflon Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-16B</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene 50mL High Speed Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-10K</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon,sterile, capped 50mL conical disposable tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>#21008-939</td>
<td>&nbsp;</td>
<tr>
<td>Phase Lock Gel Heavy, 2mL</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#2302830</td>
<td>&nbsp;</td>
<tr>
<td>Ambion RNase-free Microfuge Tubes, 2mL</td>
<td>&nbsp;</td>
<td>Ambion, Inc.</td>
<td>#AM12425</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

an improved method of rna isolation from loblolly pine (p. taeda l.) and other conifer species

Tejidos aislados de especies de coníferas, en especial los que pertenecen a la familia Pinaceae, como el pino taeda (<em&gt; Pinus taeda</em&gt; L.), contienen altas concentraciones de compuestos fenólicos y polisacáridos que interfieren con la purificación de ARN. Aislamiento de ARN de alta calidad de estas especies requiere de rigurosos procedimientos de recogida de tejidos en el campo y el empleo de un protocolo de aislamiento de ARN consta de varios pasos de extracción orgánica con el fin de aislar el ARN de calidad suficiente para microarrays y otros análisis genómico. El aislamiento de ARN de alta calidad desde el campo las muestras recogidas pino de incienso puede ser un reto, pero varias modificaciones en los tejidos y los procedimientos estándar de aislamiento de ARN en gran medida a mejorar los resultados. El grado de general de la degradación del ARN aumenta si las muestras no están debidamente recogidos y transportados desde el campo, sobre todo en gran escala de las cosechas. Total de los rendimientos de ARN se puede aumentar de manera significativa por las muestras de pulverización en un molino de nitrógeno líquido antes de la congelación de aislamiento de ARN, especialmente cuando las muestras proceden de los tejidos leñosos. Esto se debe principalmente a la presencia de agentes oxidantes, como los compuestos fenólicos y polisacáridos que están presentes en altos niveles en los extractos de los tejidos leñosos de las especies más coníferas. Si no se elimina, estos contaminantes pueden llevar a más de los principales problemas, como la degradación del ARN, que se traducen en bajos rendimientos y una muestra de la mala calidad del ARN. El arrastre de los compuestos fenólicos, así como los polisacáridos, también puede reducir o incluso eliminar por completo la actividad de la transcriptasa inversa o polimerasas otros de uso común para la síntesis de cDNA. En particular, el ARN destinado a ser utilizado como molde para la doble cadena de síntesis de ADNc en la generación de bibliotecas de ADNc de una sola hebra de síntesis de ADNc por PCR o de PCR cuantitativa, o para la síntesis de materiales de destino de microarrays deben ser de la más alta calidad si los investigadores esperan que para obtener resultados óptimos. Técnicas de aislamiento de ARN comúnmente empleados para muchas especies de plantas son a menudo insuficientes en su capacidad para eliminar estos contaminantes a partir de muestras de coníferas y por lo tanto no dan muestras de ARN total adecuado para manipulaciones posteriores. En este video se demuestra métodos para la recogida de campo de los tejidos de coníferas, a partir de la tala de un cuarenta años de edad del árbol, para la recolección de floema, xilema secundario y xilema madera de reacción. También demuestran un protocolo de aislamiento de ARN que siempre ha dado de alta calidad del ARN para su posterior manipulación enzimática.

Watch this Scientific Journal Video about An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species at JoVE.com

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Muchos tejidos de plantas, incluyendo el floema y el xilema de pino taeda (<em&gt; Pinus taeda</em&gt; L.), contienen altos niveles de compuestos fenólicos y polisacáridos que interfieren con la purificación de ARN. Esta presentación se analizan las técnicas para la cosecha de tejidos cultivadas en el campo y el aislamiento de ARN de calidad suficiente para microarrays y otros análisis genómico.

Un método mejorado para el aislamiento de ARN a partir de pino taeda ( P. taeda L.) y otras especies de coníferas

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

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Research

JoVE Journal

Biology

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

13.7K vistas.  University of Georgia (UGA). Muchos tejidos de plantas, incluyendo el floema y el xilema de pino taeda ( Pinus taeda L.), contienen altos niveles de compuestos fenólicos y polisacáridos que interfieren con la purificación de ARN. Esta presentación se analizan las técnicas para la cosecha de tejidos cultivadas en el campo y el aislamiento de ARN de calidad suficiente para microarrays y otros análisis genómico.

Video: An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Processing the Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. 
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips&trade; are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48&deg;C in high humidity conditions. After hybridization, a series of standard washes are done at 53&deg;C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

Procesamiento de los Pinus taeda PtGen2 cDNA microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

Investigación

Biología

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Aislamiento del ARN de síntesis embrionarias pez cebra y el cDNA de análisis de expresión génica

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Perfiles de metiltransferasas y Otros S-Adenosil- L-Homocisteína proteínas que se unen mediante espectrometría de masas de captura compuesto (MCP)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Aislamiento y purificación de Kinesin desde Drosophila Los embriones

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

Vampiric Aislamiento de líquido extracelular de<em&gt; Caenorhabditis elegans</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Un método mejorado para la rápida y precisa medición de rendimiento en vuelo<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

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

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

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

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

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Híbrido-Cut: un método de seccionamiento mejorada para muestras de tejido de la planta recalcitrante

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

Cinta-cáscara de estomas: Un método mejorado para la preparación de la muestra de protector de la célula

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Un protocolo mejorado de tiempo y mano de obra eficiente para el aislamiento de hepatocitos primarios en ratones

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...