Reacción de la transcriptasa inversa A su vez un calentador a 70-80 ° C y 42 ° C. Baños de agua también se puede utilizar en este paso. Si utiliza bloques de calentamiento, poner un poco de agua para que el calor es uniforme. <ol><li> Cebado reacción <ol><li> Tomar 3 g de ARN </li><li> Ajustar el volumen a 13 ml con agua libre de RNasa </li><li> Añadir 2,5 l de azar hexamer primers (2mg/ml) </li></ol></li><li> Mezclar bien e incubar a 70-80 ° C durante 8 minutos. Después, coloque en hielo durante 5 min. </li><li> Preparar cócteles RT (14,5 l / rxn): <table border="0"><tbody><tr><td> Componente </td><td> Volumen </td></tr><tr><td> Buffer 5X primer capítulo </td><td> 6 l </td></tr><tr><td> 0,1 M TDT </td><td> 3 l </td></tr><tr><td> 25X aminoallyl-Mezcla de dNTP </td><td> 1,2 l </td></tr><tr><td> Superíndice II RT (200U/μl) </td><td> 2,3 l </td></tr><tr><td> De agua </td><td> 2,0 l </td></tr></tbody></table> * De las reacciones n, preparar mezcla maestra para n 0,5 alícuotas de cóctel para asegurar que no se acabará. </li><li> Añadir 14,5 l reacciones enfriado. </li><li> Mix (no vortex) y se incuba a 42 ° C durante 3-4 horas </li><li> Las muestras pueden ser almacenadas a -20 ° C si es necesario. </li></ol> Hidrolizar ARN <ol><li> Para cada muestra, añadir 3 l de NaOH 1 N y 0,6 l de 0,5 M EDTA. (Final conc.s NaOH = 100 mm, 10 mm EDTA) </li><li> Mezclar e incubar a 65 ° C durante 10 minutos. </li><li> Neutralizar mediante la adición de 33,6 HEPES 1M l, pH = 7.0 (final conc .= 500 mm HEPES) </li><li> Las muestras pueden ser almacenadas a -20 ° C, si es necesario. </li></ol> Purificación de la reacción: Eliminación de no incorporadas aa-dUTP y aminas libres Nota: Este protocolo de purificación es una modificación del protocolo de PCRpurification Qiagen QIAquick kit. El lavado del fosfato y tampones de elución se sustituyen por los buffers Qiagen suministrados por los topes Qiagen contienen aminas libres que compiten con la reacción de acoplamiento Cy tinte. Las columnas de la mini-kit de preparación también se puede utilizar. <ol><li> Mezcla de reacción de cDNA de 300 l (o volumen de reacción 5X = 336 l) buffer PB (Qiagen incluido) y traslado a la columna QIAquick. </li><li> Colocar la columna en un tubo de recogida de 2 ml (Qiagen incluido) y se centrifuga a ~ 13.000 rpm durante 1 minuto. Tubo de extracción de vacío. </li><li> Para lavar, añadir 750 l buffer de lavado de fosfato (no de Qiagen) a la columna y centrifugar a ~ 13.000 rpm durante 1 minuto. </li><li> Vaciar el tubo de recogida y repetir el lavado de 750 l, y la centrifugación. </li><li> Vaciar el tubo de recogida y centrifugar la columna de un adicional de 1 minuto a máxima velocidad. </li><li> Transferencia de columna a un tubo de microcentrífuga de 1.5 ml y con prudencia, 30 buffer de elución l de fosfato. (Véase la sección preparación de la solución) </li><li> Incubar durante 1 minuto a temperatura ambiente. </li><li> Elución por centrifugación a ~ 13.000 rpm durante 1 minuto. </li><li> Eluir por segunda vez (con otro 30 l de tampón fosfato de elución) en el mismo tubo repitiendo los pasos 6-8. El volumen de elución final debe ser ~ 60 microlitros. </li><li> La cantidad de cDNA se puede cuantificar en este momento: ng cDNA = (dilución de los factores) (DO260) (37) (volumen total eluye de la columna) </li><li> Muestra seca en un Speed ​​Vac a 45 ° C. </li><li> Las muestras pueden ser almacenadas a -20 ° C si es necesario. </li></ol> Acoplamiento aa-cDNA de Cy Ester tinte <ol><li> Resuspender aminoallyl marcado cDNA con 10 l 50 mM bicarbonato sódico, pH = 9,0 si es necesario. (Este buffer debe ser fresca cada dos semanas) </li><li> Trabajar en un lugar oscuro, añadir la sonda a un tubo de tinte Cy apropiado. Pipeta veces arriba y abajo varias para volver a suspender el tinte. </li><li> Incubar la reacción durante 1 hora en la oscuridad a temperatura ambiente. (Incubación puede llegar hasta dos horas) </li></ol> Reacción de purificación II: La eliminación del colorante desacoplado <ol><li> A la reacción de añadir 35 l 100 mM pH 5,2 NaOAc. Mezclar bien. </li><li> Añadir 250 l (o volumen de reacción 5X = 225 l) Buffer PB (Qiagen suministrado) para cada muestra y mezclar pipeteando arriba y hacia abajo. </li><li> Coloque una columna de centrifugación QIAquick en un tubo de recogida de 2 ml (Qiagen suministrado), aplicar la muestra a la columna y centrifugar a ~ 13.000 durante 1 minuto. Tubo de extracción de vacío. </li><li> Para lavar, añadir 0,75 ml de tampón PE (Qiagen suministrado) a la columna y centrifugar a ~ 13.000 durante 1 minuto. </li><li> Repita el paso 4. </li><li> Tubo de extracción de vacío y la columna de centrifugación durante 1 minuto a velocidad máxima. </li><li> Columna a cabo en un tubo de microcentrífuga limpio de 1.5 ml y con prudencia, 30 l Buffer EB (Qiagen incluido) para el centro de la membrana de la columna. </li><li> Incubar durante 1 minuto a temperatura ambiente. </li><li> Elución por centrifugación a ~ 13.000 rpm durante 1 minuto. </li><li> Eluir por segunda vez en el mismo tubo mediante la repetición delos pasos 7-9. El volumen de eluido final debe ser ~ 60 microlitros. </li></ol> Análisis de la reacción de marcaje La cantidad de cDNA y la etiqueta se puede cuantificar en este momento: <ul><li> Medida DO 260, 550 y 650 (blanco es el agua.) Utilice el programa de microarrays nanodrop. </li><li> Calcular la pmol de incorporación colorantes y el ADN y la relación de nuc pmol / colorante con las ecuaciones que figuran a continuación: nucleótidos pmol = [DO260 volumen * (l) * 37 ng / mL * 1000 pg / ng] / 324,5 pg / pmol </li></ul> Nota: 1 DO260 = 37 ng / mL de cDNA, 324,5 pg / pmol peso molecular medio de un dNTP) pmol Cy3 OD550 = volumen * (l) / 0,15 pmol Cy5 = OD650 volumen * (l) / 0,25 nucleótidos / relación de tinte = pmol cDNA / pmol Cy tintes Si está utilizando nanodrop, que ya da pmol / l para cada tinte. Sólo tiene que multiplicar este número por el volumen para conseguir la incorporación total de tinte pmol. Nota:&gt; 150-200 pmol de incorporación de la tintura por cada muestra y una proporción de menos de 20 nucleótidos / moléculas de colorante es óptimo para las hibridaciones. <ul><li> Las muestras pueden ser almacenadas a -20 ° C en la oscuridad. <ul><li> Combine las sondas que se hibridan entre sí y seco en el speedvac a 42 ° C. </li><li> Las muestras pueden ser almacenadas a -20 ° C hasta el momento de hyb. </li></ul></li></ul> Pre-hyb diapositivas <ol><li> Lugar frente a deslizarse hacia abajo por RT 100 ml de agua en un portaobjetos limpio roja durante 3-5 minutos </li><li> Ajustar la diapositiva seca colocando boca arriba sobre un bloque de calentamiento de 100 ° C durante unos segundos - velar por la condensación de desaparecer </li><li> UV cross-link de la diapositiva a 250 mJoules (Escriba 2500 para la reticulación UV) </li><li> Diapositivas de hidratos por inmersión en el pre-calentado 42 ° C el agua. Girar 5 minutos a 700 rpm a temperatura ambiente. </li><li> Diapositivas se incuban en 50 ml frasco de Coplin que contiene pre-calentado previamente hyb (5XSSC, BSA al 1%, 0,1% SDS) durante al menos 45 minutos a 42 ° C, y la incubación puede durar más tiempo si es necesario. 50 ml antes de la hibridación buffer: 12,5 ml 20x SSC, 10 ml 5% de BSA, 0,5 ml de SDS al 10%, 27 ml de agua </li><li> Sumerja los portaobjetos en agua previamente calentada (42 ° C) y agitar un par de minutos para eliminar pre-hibridación de amortiguación. </li><li> Enjuague con isopropanol y el giro 5 min a 700 rpm, temperatura ambiente y seco para. </li></ol> Preparación de muestras para la hibridación <ol><li> Sonda resuspender en agua (el volumen se indican a continuación) <table border="0"><tbody><tr><td> Para el total de volumen. = 30μl </td><td> Para el total de volumen. = 35μl </td><td> Para el total de 40μl vol .= </td><td></td></tr><tr><td> 19,8 l de agua </td><td> 23,55 l de agua </td><td> 27,2 l de agua </td><td> </td></tr><tr><td> 1,5 l </td><td> 1,5 l </td><td> 1,5 l </td><td> 10 mg / ml de ADN de esperma de salmón (Invitrogen) </td></tr><tr><td> 1,2 l </td><td> 1,2 l </td><td> 1,2 l </td><td> 12,5 mg / ml tRNA </td></tr><tr><td> 4,5 l </td><td> 5,25 l </td><td> 6 l </td><td> 20XSSC (3X final) </td></tr><tr><td> 3 l </td><td> L 3,5 </td><td> 4 l </td><td> SDS al 1% </td></tr></tbody></table> Nota: El orden de adición de los reactivos son importantes. No preparar la mezcla principal. </li><li> Hervir durante 5 minutos, luego girar 5 minutos a 14.000 rpm. Deje enfriar durante 2 min a temperatura ambiente. </li></ol> Hibridación <ol><li> Añadir 10-12 l de 3X SSC a los pozos de la cámara de hibridación en cada borde y en el medio. </li><li> Añadir una hoja de levantador de limpieza en el área adecuada de la diapositiva y la suavidad de carga en solución de hibridación (30 -40 l). No debe ser muestra adicional sobre cualquiera de los bordes de la hoja de la cubierta. </li><li> Colocar la placa en la cámara. Apretar los tornillos y colocar en un baño de agua de 65 ° C durante la noche. No coloque la cámara directamente sobre el fondo del baño de agua. Coloque un bloque en la parte superior de la cámara hacia arriba para mantener todo en su lugar. Mantenga la tapa en el baño de agua para proteger las muestras sensibles a la luz. </li></ol> Hyb Lava <ol><li> Prepare los siguientes topes: <ul><li> 1X SSC, SDS al 0,05% </li><li> 0.06 X SSC </li></ul></li><li> Después de la cámara de incubación hibridación desprecintar. Elimina la diapositiva de la cámara, teniendo cuidado de no perturbar el cubreobjetos. </li><li> Para eliminar cubreobjetos, diapositivas sumergirse en un recipiente lleno de cálida SSC 1X, 0,05% SDS cubreobjetos tampón de lavado que enfrenta el fondo del plato. El cubreobjetos se caerá por el lado de diapositivas en movimiento a otro. </li><li> Después de que el cubreobjetos se retira, colocar la placa en un bastidor de un refrescante baño con agua tibia SDS 1XSSCm 0,05%. Manifestarse a favor de un par de minutos </li><li> Transferencia de diapositivas, además de rack para un baño de 0,06 X SSC. </li><li> Transferencia de diapositivas a otro baª de 0,06 X SSC que contiene una rejilla nueva, borrar la diapositiva sobre una toalla de papel para eliminar la mayor cantidad de SDS-tampón que contiene como sea posible antes de colocar en el baño fresco. </li><li> Agite desliza por un par de minutos. </li><li> Girar inmediatamente a temperatura ambiente durante 5 min a 700 rpm, se desliza esté listo para escanear. Almacenar en la oscuridad hasta escaneados. </li></ol>

<ol>
	<li>Hasseman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TIGR+Aminoallyl+Labeling+of+RNA+for.">TIGR Aminoallyl Labeling of RNA for.</a> Microarrays &amp; TIGR Microarray Labeled Probe Hybridization. , .</li><li>Gilbert, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TIGR+Microbial+RNA+Aminoallyl+Labeling+for+Microarrays+&amp;+Hybridization+of+probed+labels.">TIGR Microbial RNA Aminoallyl Labeling for Microarrays &amp; Hybridization of probed labels.</a> , .</li><li>Hedge, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+concise+guide+to+cDNA+Microarray+analysis-II.">A concise guide to cDNA Microarray analysis-II.</a> , .</li></ol>

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>AA-dUTP</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A0410</td>
<td>5-(3-aminoallyl)-2&#x2019;deoxyuridine-5&#x2019;-triphosphate</td>
</tr>
<tr>
<td>dNTP</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>27-2035-01</td>
<td>100 mM dNTP Set PCR grade</td>
</tr>
<tr>
<td>Random Hexamer primers</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>27-2166-01</td>
<td>3mg/mL</td>
</tr>
<tr>
<td>SuperScript III RT</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>80800444</td>
<td>200U/&#x03BC;L</td>
</tr>
<tr>
<td>CyDye&#x2122;</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>RPN5661</td>
<td>Post-Labelling Reactive Dye Pack</td>
</tr>
<tr>
<td>QIAquick</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28104</td>
<td>PCR Purification kit</td>
</tr>
<tr>
<td>1M K2HPO4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1M KH2PO4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1M KPO4</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To make a 1M Phosphate buffer (KPO4, pH 8.5-8.7) combine:
9.5 mL 1M K2HPO4 and 0.5 ml 1M KH2PO4
</td>
</tr>
<tr>
<td>Phosphate wash buffer</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For 100 mL Phosphate wash buffer (5 mM KPO4, pH 8.0, 80% EtOH) mix: 0.5 ml 1 M KPO4 pH 8.5 + 15.25 mL MilliQ water + 84.25 mL 95% ethanol. Wash buffer will be slightly cloudy.
** IMPORTANT: Phosphate wash buffer should be prepared daily.</td>
</tr>
<tr>
<td>Phosphate elution buffer</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Diluting 1 M KPO4, pH 8.5 to 4 mM with sterile water</td>
</tr>
<tr>
<td>Sodium Carbonate Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>(Na2CO3): 0.5M, pH 9.0. Dissolve 4.2 g NaHCO3 in 80 mL of sterile water and adjust pH to 9.0 with 10 N NaOH; bring volume up to 100 mL with sterile water. To make a 50 mM solution for the dye coupling reaction dilute 1:10 with water.
Note: Carbonate buffer changes composition over time; make it fresh every couple of weeks to a month.</td>
</tr>		</tbody>
		</table>
		</div>

bacterial gene expression analysis using microarrays

Watch this Scientific Journal Video about Bacterial Gene Expression Analysis Using Microarrays at JoVE.com

Bacterial Gene Expression Analysis Using Microarrays

Gen bacteriano y Análisis de Expresión El uso de microarrays

bacterial-gene-expression-analysis-using-microarrays

Research

JoVE Journal

Biology

8.7K vistas.  University of California Santa Cruz - UCSC. 

Video: Bacterial Gene Expression Analysis Using Microarrays

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Investigación

Biología

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

Infección por el virus vaccinia y Análisis temporal de la expresión génica del virus: Parte 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

Infección por el virus vaccinia y Análisis temporal de la expresión génica del virus: Parte 2

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Infección por el virus vaccinia y Análisis temporal de la expresión génica del virus: Parte 3

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 ...

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 ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilizando el sistema de electroporación Gene Emisores MXcell para transfectar células primarias con alta eficiencia

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

El uso de un contador de células automatizado para simplificar estudios de expresión génica: siRNA Derribo de IL-4 la expresión génica en células dependientes Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

Análisis de la expresión génica en el barrenador esmeralda del fresno ( Agrilus planipennis) Utilizando Real-PCR cuantitativa en tiempo

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

Todo el genoma análisis de Aminoacilación (de carga) Niveles de tRNA utilizando microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...