El trabajo fue apoyado en parte por el Instituto Nacional de Abuso de Drogas del Centro (P50 DA 011.806) y Ejército de Estados Unidos Departamento de Defensa (W81XWH-07-1-0183).

1. La impresión de microarrays miARN personalizado <ol><li> Imprimir los microarrays diapositivas con los servicios básicos de microarrays instalaciones de una universidad o una empresa. La calidad de fabricación de microarrays es uno de los factores más críticos para el éxito de un experimento de microarrays. Pruebe algunos de los servicios si es posible. Lo ideal sería imprimir 50-100 diapositivas a la vez, y todas las diapositivas se ven idénticas, con buena separación, los puntos individuales. </li><li> Para el apoyo de microarrays, que el uso de las diapositivas GAPS II recubiertos. </li><li> Para las sondas de miRNA, se utiliza el nCode Multi-Especies miARN microarrays sonda Set V2. </li><li> Disolver todos los oligonucleótidos en 3 x SSC a 10 M y cuádruplemente imprimirlas en las diapositivas. Fijar las diapositivas por la irradiación ultravioleta de acuerdo a las instrucciones de las diapositivas GAAPS II. Etiqueta de la diapositiva con un lápiz de diamante para marcar el área y el lado con las sondas impreso. Almacenar a 22 ° C. </li></ol> 2. Preparación de la muestra <ol><li> Cualquier métodoo reactivo se puede utilizar para aislar ARN, mientras que conserva los pequeños RNAs. Por lo general, el uso Trizol (Invitrogen) para extraer el ARN total, con la cantidad y calidad satisfactoria de preparaciones de ARN, según lo medido por un 260 nm y 280 nm A lecturas. </li><li> Para el ARN de etiquetado, mezcla de aproximadamente 25 g de ARN total con 0,5 mg de 5&#39;-UCP-DY547-3 &quot;6 en 1 x tampón (50 mM HEPES, pH 7,8, 20 mM MgCl 2, 10 mg / ml de BSA, el 10% DMSO) que contiene aproximadamente 20 unidades de T4 ARN ligasa 1, suplementado con 10 mM TDT ~ ~ 0.02-0.03 y volumen final de la 10 x T4 ARN ligasa un buffer de ATP. </li><li> Permitir el etiquetado de proceder en el hielo dentro de un refrigerador durante 2-24 horas. Precipitar el ARN con acetato de sodio 0,3 M y 3 volúmenes de etanol. Para la ligadura de eficiencia y la precipitación, se disuelven en el ARN total de más de 2 mg / ml, y mantener el volumen total de la ligadura de ~ 20 l. <ol><li> Si es menor de ARN está disponible, la cantidad de ARN de entrada y 5&#39;-UCP-DY547-3 &#39;puede ser reducido. &lt;/ Li&gt; <li> Si el ARN es muy diluida, añadir soporte como tRNA de levadura para ayudar en la precipitación. </li></ol></li><li> Combine y la etiqueta de un total de 1 mg de oligonucleótidos de ADN de referencia correspondiente a un subconjunto de miRNAs de mamíferos utilizando la Ulysis Alexa Fluor 647 Kit nucleicos Etiquetado ácido 6 como un control para el proceso de hibridación. Purificar el ADN marcado con una columna Centrisep y guardar en la oscuridad a -20 ° C. </li></ol> 3. Microarrays de hibridación <ol><li> Para utilizar las diapositivas, por primera vez, antes de la hibridación de las diapositivas de una solución de filtrado de 3 x SSC, SDS 0,1%, 0,2% BSA durante 30-60 min a 37 ° C. Sumergirlos en agua unas cuantas veces, y luego en isopropanol. Secar los portaobjetos al uso de una centrífuga con un adaptador para diapositivas a 100 g por 5 min a 22 ° C. </li></ol> Nota: Usamos Corning cámaras de hibridación de microarrays y Erie Scientific mSeries de Lifterslips para llevar a cabo la hibridación de microarrays. <ol start="2"><li> Enjuague la lifterslips en el agua, y luego en etanol, antes del secado en el aire. Coloque una diapositiva dentro de una base de la cámara de microarrays de hibridación, y un lifterslip en la parte superior de la diapositiva. El lifterslip cubrirá el área de la sonda, con sus franjas blancas en contacto con la diapositiva. </li><li> En la oscuridad, la desaceleración del precipitado, ARN marcado. Lavar una vez con etanol al 70%, y el aire seco de la pastilla. La pastilla debe ser de color rojizo. </li><li> Prepare una solución de hibridación que contenía 400 mM Na 2 HPO 4 pH 7,0, el 0,8% de BSA, 5% SDS, 12% de formamida 6 con 1/15-1/100 l de la purificada, con la etiqueta de ADN de referencia (2,4) por cada muestra de ARN. La cantidad de ADN de referencia añadido depende de la cantidad de diferentes oligonucleótidos están etiquetados (2,4). Si hay más de 100, entonces una menor dilución que se necesita. </li><li> Disolver el precipitado de ARN bien con aproximadamente 60 l de la solución de hibridación. </li><li> Añadir 20 l de agua para cada uno de los pozos de humidificación en la base de Corning cámara de hibridación de microarrays. </li><li> Añadir tque mezcla de ARN y el ADN marcado referencia a la diapositiva. Utilice una punta de pipeta fina, toque suavemente el borde de la lifterslip, y permitir que la solución penetre en el espacio entre el lifterslip y deslizarse a través de la succión. </li><li> Coloque la cámara de hibridación cubierta sobre la base, luego sellar la cámara con clips metálicos. </li><li> Ponga toda la cámara interior de la bolsa de plástico que viene con el cartucho de cámara de Corning. </li><li> Coloque la cinta de la cámara (s) dentro de un recipiente con una toalla de papel humedecida, cubra el recipiente con papel plástico y se coloca dentro de 37 ° C húmedo, incubadora de cultivo de células de ~ 24 horas. Estas precauciones (3.6, 3.9, 3.10) asegurarse de que la solución de hibridación no se seque durante la incubación. </li></ol> 4. Después de la hibridación de procesamiento <ol><li> Desmontar los casetes de la cámara de uno por uno. Sumerja una diapositiva y su lifterslip en 2 x SSC a 22 ° C. El lifterslip caerá naturalmente fuera de la diapositiva. Colocarlo en 0,8 x SSC. Lavarlas diapositivas en 0,8 x SSC dos veces, tres veces en 0,4 x SSC, 1-2 minutos en total. Secar los portaobjetos al uso de una centrífuga con un adaptador para diapositivas. Lave la lifterslips con agua y ahorrar para su reutilización posterior. </li><li> Escaneo de las diapositivas con cualquier escáner adecuado, por ejemplo, la Perkin Elmer 5000 o ScanArray Molecular Devices AXON GenePix 4000B escáner de microarrays. Exploración en longitudes de onda cercana a 547 nm y 647 para obtener los archivos de imagen correspondiente. </li><li> Utilizar programas como BlueFuse para cuantificar la intensidad de los píxeles de la entrada, los archivos de imagen de 4,2). Inspeccione los puntos individuales con el programa para excluir zonas anormales en los microarrays de mayor consideración. Estas manchas anormales suelen surgir de la impresión de diapositivas o diapositivas pobre manejo después de la hibridación. </li><li> Utilizar programas como GeneSpring y Excel para el análisis y presentación de datos. Consideraciones generales para la normalización de los datos de microarrays se aplican, lo que está fuera del alcance de este documento. </li><li> Una vez que las señales satisfactorios se obtienen after 4.3), tira de las diapositivas de microarrays para la reutilización 7. Enjuague los portaobjetos en agua y luego sumergirse en la pre-calentado, 1 mM NaOH y 0,1 x SSC en un plato de tinción a 62 ° C durante 10-20 minutos. Repita la incubación una vez. Lave el C. desliza un par de veces en el agua hasta por 60 minutos con agitación suave a 22 ° Secar los portaobjetos utilizando una centrifugadora, y almacenar a 22 ° C. </li><li> Para utilizar las diapositivas regenerado, no es necesario para pre-hibridación de nuevo. Simplemente lavarlos en agua, seguido de isopropanol, y seca las diapositivas justo antes de la hibridación. </li></ol> 5. Los resultados representativos: En gran medida hemos seguido los procedimientos descritos en el perfil de expresión de miRNA mundial en miles de muestras, es decir, ARN aislado de las muestras de los peces cebra que bajo muchas condiciones diferentes. La figura 1 muestra una imagen escaneada de un microarray de demostrar señales de hibridación muy precisas y fuertes en el diapositivas. El correlati Pearsonen los coeficientes entre repeticiones técnica de hibridación de microarrays se ~ 0.99 7, lo que indica una excelente reproducibilidad. <img alt="Figura 1" src="/files/ftp_upload/3250/3250fig1.jpg" /> Figura 1 Imagen compuesta de una diapositiva escaneada miARN microarrays después de la hibridación. Manchas rojas resultado de hibridaciones por el ADN de referencia, los puntos verdes de la muestra DY547-RNA marcado, mientras que las manchas amarillas eran de hibridaciones tanto por el ADN y el ARN de las mismas sondas.

<ol>
	<li>Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functions+of+animal+microRNAs.">The functions of animal microRNAs.</a> Nature. 431, 350-355 (2004).</li><li>Friedman, R. C., Farh, K. K., Burge, C. B., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92-105 (2009).</li><li>Chekulaeva, M., Filipowicz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+miRNA-mediated+post-transcriptional+regulation+in+animal+cells.">Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells.</a> Curr. Opin. Cell Biol. 21, 452-460 (2009).</li><li>Farazi, T. A., Spitzer, J. I., Morozov, P., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+in+human+cancer.">miRNAs in human cancer.</a> J. Pathol. 223, 102-115 (2011).</li><li>Small, E. M., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pervasive+roles+of+microRNAs+in+cardiovascular+biology.">Pervasive roles of microRNAs in cardiovascular biology.</a> Nature. 469, 336-342 (2011).</li><li>Thomson, J. M., Parker, J., Perou, C. M., Hammond, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+custom+microarray+platform+for+analysis+of+microRNA+gene+expression.">A custom microarray platform for analysis of microRNA gene expression.</a> Nat. Methods. 1, 47-53 (2004).</li><li>Zhang, X., Xu, W., Tan, J., Zeng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stripping+custom+microRNA+microarrays+and+the+lessons+learned+about+probe:slide+interactions.">Stripping custom microRNA microarrays and the lessons learned about probe:slide interactions.</a> Anal. Biochem. 386, 222-227 (2009).</li><li>Griffiths-Jones, S., Saini, H. K., van Dongen, S., Enright, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+tools+for+microRNA+genomics.">miRBase: tools for microRNA genomics.</a> Nucl. Acids. Res. 36, D154-D158 (2008).</li><li>Landgraf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mammalian+microRNA+expression+atlas+based+on+small+RNA+library+sequencing.">A mammalian microRNA expression atlas based on small RNA library sequencing.</a> Cell. 129, 1401-1414 (2007).</li><li>Chiang, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+microRNAs:+experimental+evaluation+of+novel+and+previously+annotated+genes.">Mammalian microRNAs: experimental evaluation of novel and previously annotated genes.</a> Genes. Dev. 24, 992-1009 (2010).</li></ol>

No hay conflictos de interés declarado.

A pesar de los avances recientes en profundidad las tecnologías de secuenciación, microarrays sigue siendo una opción viable de alto rendimiento para el análisis de ADN y ARN. En comparación con la secuenciación de profundidad, los experimentos de microarrays son más baratos, y un laboratorio típico de la biología molecular puede realizar la mayoría de los experimentos y análisis de datos in-house, que permite flexibilidad y ahorro de tiempo. En el futuro, es probable microarrays bien adaptado para interrogar...

<table border="1"><tbody><tr><th> Artículos </th><th> Vendedor </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> NCode Multi-Especies miARN microarrays sonda Set V2 </td><td> Invitrogen </td><td> MIRMPS201 </td><td> Diseñado en base a la versión 9.0 miRBase (octubre de 2006). Contiene ~ 1140 sin modificar, 34-44 nt largo oligonucleótidos como sondas de gusano, la mosca, el pez cebra, ratón, rata, y miRNAs humanos, y una serie de sondas de control interno, tales como snoRNAs. Las sondas de miRNA son dobletes de las secuencias complementarias de miRNAs maduros, por lo tanto, el tamaño de ~ 44 nt. Para el análisis se puede centrar en los miRNAs de un genoma particular (s) de interés. </td></tr><tr><td> Trizol </td><td> Invitrogen </td><td> 15596018 </td><td> También hemos utilizado enriquecido, pequeña fracción de ARN para el etiquetado, aunque el total de muestras de ARN son más rápidos y más fáciles de preparar y cuantificar y adecuado para las aplicaciones posteriores tales como el análisis de ARNm. </td></tr><tr><td> T4 RNA ligase 1 </td><td> Nueva Inglaterra Biolabs </td><td> M0204L </td><td></td></tr><tr><td> Ulysis Alexa Fluor 647 Kit de etiquetado ácido nucleico </td><td> Invitrogen </td><td> U21660 </td><td> Este kit o productos similares, puede ser utilizada para etiquetar experimental muestras de ARN o un ARN de control (en lugar de ADN control) también. </td></tr><tr><td> 5&#39;-UCP-DY547-3 &#39; </td><td> Dharmacon </td><td> Una medida que se </td><td> Pequeña fracción de ARN pueden ser etiquetados de manera similar por la ligadura. </td></tr><tr><td> Centrisep columnas </td><td> Princeton separaciones </td><td> CS-901 </td><td></td></tr><tr><td> GAPSII recubierto de diapositivas </td><td> Corning </td><td> 40004 </td><td> Otros tipos de diapositivas puede también ser utilizado. </td></tr><tr><td> Microarrays de cámaras de hibridación </td><td> Corning </td><td> 2551 o 40080 </td><td> Otros tipos de cámaras de hibridación y cubreobjetos también debería funcionar. Uso de máquinas de hibridación comercialmente disponibles puede reducir significativamente el tiempo de hibridación, por ejemplo, a ~ 2 horas. </td></tr><tr><td> Lifterslips </td><td> ªERMO / Erie Científico </td><td> 25X60I-M5439-001-LS </td><td></td></tr><tr><td> BlueFuse </td><td> BlueGenome </td><td></td><td></td></tr><tr><td> GeneSpring </td><td> Agilent </td><td></td><td></td></tr></tbody></table>

performing custom microrna microarray experiments

microARN (miRNA) son una gran familia de ~ 22 nucleótidos (nt) de longitud moléculas de ARN que se expresan ampliamente en las células eucariotas 1. Genomas complejos codifican por lo menos cientos de miRNAs, que sobre todo inhibir la expresión de un gran número de genes diana transcripcional después de 2, 3. miRNAs control de una amplia gama de procesos biológicos 1. Además, la expresión alterada miRNA se ha asociado con enfermedades humanas como el cáncer, y los miRNAs pueden servir como biomarcadores de enfermedades y el pronóstico 4, 5. Es importante, por lo tanto, para entender la expresión y función de los miRNAs en diferentes condiciones. Tres enfoques principales han sido empleados para el perfil de expresión de miARN: PCR en tiempo real, de microarrays, secuenciación y profundidad. La técnica de los microarrays de miRNA tiene la ventaja de ser de alto rendimiento, en general, menos costoso, y la mayoría de las medidas experimentales y el análisis puede ser carrIED en un laboratorio de biología molecular en la mayoría de las universidades, escuelas médicas y hospitales asociados. Aquí se describe un método para realizar experimentos de microarrays de encargo miRNA. Una sonda conjunto miRNA se imprimirán en láminas de vidrio para producir microarrays miRNA. El ARN es aislado utilizando un método o reactivo que preserva las especies pequeñas de ARN, y marcado con un colorante fluorescente. Como control, los oligonucleótidos de ADN de referencia que corresponde a un subconjunto de miRNAs están marcados con un colorante fluorescente diferente. El ADN de referencia servirá para demostrar la calidad de la diapositiva y la hibridación y también se utiliza para la normalización de datos. El ARN y el ADN se mezclan y se hibridó con una diapositiva que contiene sondas de microarrays para la mayoría de los miRNAs en la base de datos. Después del lavado, la diapositiva se escanea para obtener imágenes y cuantificar la intensidad de los puntos individuales. Estas señales en bruto será procesada y analizada, los datos de expresión de los miRNAs correspondiente. MicroArray diapositivas se puede eliminar y se regenera para reducir el costo de microarrays y de mejorar la coherencia de experimentos de microarrays. Los mismos principios y procedimientos son aplicables a otros tipos de experimentos de microarrays personalizados.

Watch this Scientific Journal Video about Performing Custom MicroRNA Microarray Experiments at JoVE.com

Performing Custom MicroRNA Microarray Experiments

Un procedimiento sencillo de llevar a cabo experimentos de microarrays de encargo de microARN se describe. Los pasos incluyen el aislamiento de ARN, el ARN y el ADN de referencia el etiquetado, la hibridación de las muestras de microarrays, el escaneado de los microarrays, cuantificar y analizar las señales de hibridación.

Realización de experimentos de microarrays personalizados microARN

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1.  Complex genomes encode ...

performing-custom-microrna-microarray-experiments

Research

JoVE Journal

Biology

19.4K vistas.  University of Minnesota. Un procedimiento sencillo de llevar a cabo experimentos de microarrays de encargo de microARN se describe. Los pasos incluyen el aislamiento de ARN, el ARN y el ADN de referencia el etiquetado, la hibridación de las muestras de microarrays, el escaneado de los microarrays, cuantificar y analizar las señales de hibridación.

Video: Performing Custom MicroRNA Microarray Experiments

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae ascósporas de producción y de recolección de experimentos de microarrays.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

Investigación

Biología

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Preparación de la bicapa artificial para experimentos de electrofisiología

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

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 ...

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Gene mundial y Análisis de Expresión utilizando una plataforma de microarrays de oligonucleótidos de pez cebra

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

El análisis de microarrays para Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

Genoma Edición con CompoZr nucleasas de dedos de zinc personalizados (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

MicroRNA<em&gt; In situ</em&gt; La hibridación de los tejidos del riñón y fijados con formalina

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and cardiovascular disease, have been shown to activate tissue and/or cell-specific miRNA expression associated with disease progression. The inhibition of miRNA expression offers the potential for a therapeutic intervention. However, traditional approaches to inhibit miRNAs, employing antagomir oligonucleotides, affect specific miRNA functions upon global delivery. Herein, we present a protocol for the in vivo cardio-specific inhibition of the miR-181 family in a rat model. A miRNA-sponge construct is designed to include 10 repeated anti-miR-181 binding sequences. The cardio-specific &#945;-MHC promoter is cloned into the pEGFP backbone to drive the cardio-specific miR-181 miRNA-sponge expression. To create a stable cell line expressing the miR-181-sponge, myoblast H9c2 cells are transfected with the &#945;-MHC-EGFP-miR-181-sponge construct and sorted by fluorescence-activated cell sorting (FACs) into GFP positive H9c2 cells which are cultured with neomycin (G418). Following stable growth in neomycin, monoclonal cell populations are established by additional FACs and single cell cloning. The resulting myoblast H9c2-miR-181-sponge-GFP cells exhibit a loss of function of miR-181 family members as assessed through the increased expression of miR-181 target proteins and compared to H9c2 cells expressing a scramble non-functional sponge. In addition, we develop a nanovector for the systemic delivery of the miR-181-sponge construct by complexing positively charged liposomal nanoparticles and negatively charged miR-181-sponge plasmids. In vivo imaging of GFP reveals that multiple tail vein injections of a nanovector over a three-week period are able to promote a significant expression of the miR-181-sponge in a cardio-specific manner. Importantly, a loss of miR-181 function is observed in the heart tissue but not in the kidney or the liver. The miRNA-sponge is a powerful method to inhibit tissue-specific miRNA expression. Driving the miRNA-sponge expression from a tissue-specific promoter provides specificity for the miRNA inhibition, which can be confined to a targeted organ or tissue. Furthermore, combining nanovector and miRNA-sponge technologies permits an effective delivery and tissue-specific miRNA inhibition in vivo.

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Tissue-specific microRNA inhibition is a technology that is underdeveloped in the microRNA field. Herein, we describe a protocol to successfully inhibit the miR-181 microRNA family in myoblast cells from the heart. Nanovector technology is used to deliver a microRNA sponge that demonstrates significant in vivo cardio-specific miR-181 family inhibition.

En Vivo Nanovector entrega de una esponja de microARN específicos de corazón

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and ...

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30%&#160;of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic.
The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment.
The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment.

Herramienta de edición de genes CRISPR para el análisis de redes de clústeres de microARN

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published ...

Performing Muscle Cryoinjury on a Zebrafish

For performing the myomere cryoinjury on zebrafish, use a custom manufactured stainless steel cryoprobe. To avoid frostbite while handling the probe during the procedure, insert the probe handle into a pipette tip and secure it by wrapping it with tape. Before beginning with the cryoinjury procedure, prepare a beaker for the anesthesia working solution, a spoon for handling the fish, a moist sponge, and a tank with system water for post-procedure recovery of the fish.Prepare the anesthesia stock solution following the steps shown on the screen. Then add four milliliters of the prepared stock solution to 100 milliliters of system water in a beaker to prepare the tricaine working solution. To begin the cryoinjury procedure, immerse the probe in liquid nitrogen for a minimum of three minutes.Wet the sponge in system water and place it on a flat surface. Then one to two minutes after transferring a single adult zebrafish into the tricaine working solution, confirm its unresponsiveness by touching the fish gently with the spoon. Place the anesthetized fish onto the wet sponge.Locate the caudal peduncle posterior to the anal fin and anterior to the caudal fin. At this stage, remove the cryoprobe from the liquid nitrogen. Shake the probe gently to ensure no residual liquid nitrogen remains on the tip.Place the edge of the spatula perpendicular to the body on the caudal peduncle. Keep the probe in this position for six seconds without applying pressure, before releasing the cryoprobe from the tissue. Then transfer the fish into the system water in the recovery tank.Monitor the fish while it resumes breathing and swimming after waking up from the anesthesia. If opercular movements do not occur after 30 seconds, stimulate the fish by pipetting system water into the gills until the animal initiates respiration by itself. Video recording of control and cryoinjured fish at one day post-injury or DPCI revealed that although the cryoinjured fish swam less actively, they did not display any abnormal movements, such as swirling, convolution, or reduced equilibrium.In the husbandry system, their position in the tank and food intake was similar to those of the uninjured fish. As observed at 5 DPCI, normal behavior was still persistent indicating the cryoinjury procedure did not severely affect the wellbeing of the animals.