Réaction de la transcriptase Tourner bloc chauffant à 70-80 ° C et 42 ° C. Bains d&#39;eau peut également être utilisé dans cette étape. Si l&#39;aide de blocs de chauffage, mettre un peu d&#39;eau afin que la chaleur est uniforme. <ol><li> La réaction d&#39;amorçage <ol><li> Prenez 3 pg d&#39;ARN </li><li> Réglez le volume à 13 ul avec de l&#39;eau RNase </li><li> Ajouter 2,5 pi de hasard hexamère amorces (2mg/mL) </li></ol></li><li> Bien mélanger et incuber à 70-80 ° C pendant 8 minutes. Ensuite, placer sur la glace pendant 5 min. </li><li> Préparer un cocktail RT (14,5 l / Rxn): <table border="0"><tbody><tr><td> Composante </td><td> Volume </td></tr><tr><td> 5X tampon First Strand </td><td> 6 pl </td></tr><tr><td> 0,1 M de DTT </td><td> 3 pl </td></tr><tr><td> 25X aminoallyl-mélange de dNTP </td><td> 1,2 ul </td></tr><tr><td> SuperScript II RT (200U/μl) </td><td> 2,3 ul </td></tr><tr><td> Eau </td><td> 2,0 ul </td></tr></tbody></table> * Pour les réactions n, préparer Mix Master 0.5 pour n aliquotes de cocktail pour vous assurer que vous ne manquerez pas. </li><li> Ajouter 14,5 ul de réactions refroidi. </li><li> Mélanger (ne pas vortexer) et incuber à 42 ° C pendant 3-4 heures </li><li> Les échantillons peuvent être conservés à -20 ° C si nécessaire. </li></ol> Hydrolyser l&#39;ARN <ol><li> Pour chaque échantillon, ajouter 3 l de NaOH 1 N et 0,6 ul de 0,5 M d&#39;EDTA. (Final conc.s = 100mm NaOH, 10 mM EDTA) </li><li> Mélanger et incuber à 65 ° C pendant 10 minutes. </li><li> Neutraliser en ajoutant 33,6 ul de HEPES 1M, pH = 7,0 (finale conc .= 500mm HEPES) </li><li> Les échantillons peuvent être conservés à -20 ° C, si nécessaire. </li></ol> Purification de réaction: Suppression des non constituée en société AA-dUTP et amines libres Remarque: Ce protocole de purification est modifié par rapport au protocole de Qiagen QIAquick PCRpurification kit. Le lavage de phosphate et de tampons d&#39;élution sont substitués aux tampons Qiagen fourni parce que les tampons Qiagen contiennent des amines libres en concurrence avec la réaction de couplage Cy colorant. Les colonnes de la trousse de mini-préparation peut également être utilisé. <ol><li> Mélanger la réaction d&#39;ADNc avec 300 pl (ou volume de réaction 5X = 336 ul) de tampon PB (Qiagen fourni) et le transfert à la colonne QIAquick. </li><li> Placer la colonne dans un tube collecteur de 2 ml (Qiagen fourni) et centrifuger à 13000 rpm ~ 1 minute. Tube de collection vide. </li><li> Pour laver, ajouter 750 pi de tampon phosphate de lavage (pas de Qiagen) à la colonne et centrifuger à 13000 rpm ~ 1 minute. </li><li> Vider le tube de collecte et de répéter le lavage 750 ul et l&#39;étape de centrifugation. </li><li> Vider le tube de collecte et centrifuger la colonne 1 minute supplémentaire à vitesse maximale. </li><li> Transfert de colonne à une nouvelle tube de 1,5 ml et centrifuger ajouter prudemment 30 pi de tampon d&#39;élution phosphate. (Voir la section préparation de la solution) </li><li> Incuber pendant 1 minute à température ambiante. </li><li> Eluer par centrifugation à 13000 rpm ~ 1 minute. </li><li> Eluer une seconde fois (avec un autre 30 ul de tampon d&#39;élution phosphate) dans le même tube en répétant les étapes 6-8. Le volume d&#39;élution finale doit être ~ 60 pl. </li><li> Le montant de l&#39;ADNc peuvent être quantifiés à ce stade: ng d&#39;ADNc = (facteur de dilution) (OD260) (37) (volume total élué de la colonne) </li><li> L&#39;échantillon sec dans un Speed ​​Vac à 45 ° C. </li><li> Les échantillons peuvent être conservés à -20 ° C si nécessaire. </li></ol> Couplage aa-ADNc à Cy Ester Dye <ol><li> Resuspendre aminoallyl-ADNc marqué avec 10 pl 50 mM de Na-bicarbonate de pH = 9,0 si nécessaire. (Ce tampon doit être frais tous les deux semaines) </li><li> Travailler dans un endroit sombre, ajouter la sonde à un tube de colorant Cy approprié. Pipet fois monter et descendre plusieurs resuspendre le colorant. </li><li> Incuber la réaction pendant 1 heure dans le noir à température ambiante. (Incubation peut aller jusqu&#39;à deux heures) </li></ol> Purification Réaction II: Prélèvement de colorant découplé <ol><li> Pour la réaction, ajouter 35 ul de 100 mM pH 5,2 NaOAc. Mélanger soigneusement. </li><li> Ajouter 250 pi (ou volume de réaction 5X = 225 ul) Tampon PB (Qiagen fourni) pour chaque échantillon et mélanger par pipetage de haut en bas. </li><li> Placer une colonne spin QIAquick dans un tube collecteur de 2 ml (Qiagen fourni), appliquer l&#39;échantillon à la colonne, et centrifuger à 13000 ~ 1 minute. Tube de collection vide. </li><li> Pour laver, ajouter 0,75 ml de tampon PE (Qiagen fourni) à la colonne et centrifuger à 13000 ~ 1 minute. </li><li> Répétez l&#39;étape 4. </li><li> Tube de prélèvement vides et la colonne de centrifugation pour un supplément de 1 minute à vitesse maximale. </li><li> Colonne de la Place dans un endroit propre tube de 1,5 ml et centrifuger, prudemment, 30 pi de tampon EB (Qiagen fourni) pour le centre de la membrane de la colonne. </li><li> Incuber pendant 1 minute à température ambiante. </li><li> Eluer par centrifugation à 13000 rpm ~ 1 minute. </li><li> Eluer une seconde fois dans le même tube en répétantétapes 7-9. Le volume final doit être élué ~ 60 pl. </li></ol> Analyse de la réaction de marquage Le montant de l&#39;ADNc et l&#39;étiquette peuvent être quantifiés à ce stade: <ul><li> Mesure OD 260, 550 et 650 (blanc est l&#39;eau.) Utilisez le programme biopuces Nanodrop. </li><li> Calculer la pmol d&#39;incorporation des colorants et l&#39;ADN et nuc pmol / colorant rapport avec des équations ci-dessous: nucléotides pmol = [OD260 volume * (ul) * 37 ng / uL * 1000 pg / ng] / 324,5 pg / pmol </li></ul> Note: 1 OD260 = 37 ng / uL d&#39;ADNc; 324,5 pg / pmol poids moléculaire moyen d&#39;un dNTP) pmol Cy3 = OD550 volume * (ul) / 0,15 pmol Cy5 = OD650 volume * (ul) / 0,25 nucléotides / ratio de teinture = pmol ADNc / pmol Cy colorants Si vous utilisez Nanodrop, il donne déjà pmol / ul pour chaque colorant. Il vous suffit de multiplier ce nombre par le volume pour obtenir l&#39;incorporation totale de teinture pmol. Note:&gt; 150-200 pmol d&#39;incorporation de colorants par échantillon et un ratio de moins de 20 nucléotides / molécules de colorant est optimal pour les hybridations. <ul><li> Les échantillons peuvent être conservés à -20 ° C à la noirceur. <ul><li> Combinez des sondes à hybrider ensemble et sèche dans speedvac à 42 ° C. </li><li> Les échantillons peuvent être conservés à -20 ° C jusqu&#39;au moment de hyb. </li></ul></li></ul> Pré-hyb diapositives <ol><li> Placer la lame vers le bas le visage plus RT 100 ml d&#39;eau dans un support de diapositives rouges nettoyage pendant 3-5 minutes </li><li> Claquement sec de la diapositive en plaçant face visible sur une 100 ° C bloc chauffant pendant quelques secondes - montre pour la condensation à disparaître </li><li> UV réticuler la diapositive à 250 Mjoules (Entrez le réticulant 2500 à UV) </li><li> Hydratez diapositives par trempage dans l&#39;eau pré-chauffée à 42 ° C. Spin 5 min à 700 rpm à température ambiante. </li><li> Incuber dans 50ml jarre de Coplin contenant préchauffé pré-hyb (5XSSC, 1% de BSA, 0,1% SDS) pour au moins 45 minutes @ 42 ° C, et l&#39;incubation peut aller plus si nécessaire. 50 ml de tampon de pré-hyb: 12,5 mL 20x SSC, 10 ml de 5% de BSA, 0,5 ml SDS à 10%, 27 ml d&#39;eau </li><li> Plongez dans les diapositives pré-chauffé de l&#39;eau (42 ° C) et agiter quelques minutes pour enlever les pré-hyb tampon. </li><li> Rincer dans de l&#39;isopropanol et de spin 5 min à 700 rpm, la température ambiante, à sécher. </li></ol> Préparation des échantillons pour l&#39;hybridation <ol><li> Sonde Remettre en suspension dans l&#39;eau (les volumes sont donnés ci-dessous) <table border="0"><tbody><tr><td> Pour volume total. = 30μl </td><td> Pour volume total. = 35μl </td><td> Pour volume total 40μl .= </td><td></td></tr><tr><td> 19,8 ul d&#39;eau </td><td> 23,55 ul d&#39;eau </td><td> 27,2 ul d&#39;eau </td><td> </td></tr><tr><td> 1,5 pl </td><td> 1,5 pl </td><td> 1,5 pl </td><td> 10 mg / ml d&#39;ADN de sperme de saumon (Invitrogen) </td></tr><tr><td> 1,2 ul </td><td> 1,2 ul </td><td> 1,2 ul </td><td> 12,5 mg / ml d&#39;ARNt </td></tr><tr><td> 4,5 ul </td><td> 5,25 ul </td><td> 6 pl </td><td> 20XSSC (3X final) </td></tr><tr><td> 3 pl </td><td> 3,5 ul </td><td> 4 pl </td><td> SDS à 1% </td></tr></tbody></table> Note: L&#39;ordre d&#39;addition des réactifs sont importants. Ne pas préparer mélange maître. </li><li> Faire bouillir pendant 5 minutes, puis tourner 5 minutes à 14000 rpm. Refroidir pendant 2 min à température ambiante. </li></ol> Hybridation <ol><li> Ajouter 10-12 ul de 3X SSC pour les puits de la chambre d&#39;hybridation sur chaque bord et au milieu. </li><li> Ajouter un bordereau de lifter propre à la zone appropriée de la lame et en douceur de charge dans la solution d&#39;hybridation (30 -40 ul). Il devrait s&#39;agir d&#39;un échantillon supplémentaire sur chaque bord de la lamelle. </li><li> Placer la lame dans la chambre. Serrer les vis et les placer dans un bain d&#39;eau à 65 ° C pendant la nuit. Ne pas placer la chambre directement sur le fond du bain-marie. Placer un bloc sur le dessus de la chambre supérieure pour tenir tout en place. Gardez le couvercle sur le bain d&#39;eau pour protéger les échantillons sensibles à la lumière. </li></ol> Lave Hyb <ol><li> Préparer les tampons suivants: <ul><li> 1X SSC, SDS à 0,05% </li><li> 0,06 X SSC </li></ul></li><li> Après la chambre d&#39;hybridation d&#39;incubation desceller. Retirer la lame de la chambre, en prenant soin de ne pas déranger la lamelle. </li><li> Pour retirer des lamelles, des diapositives plonger dans un plat contenant 1X SSC chaude, 0,05% SDS lamelle tampon de lavage face au fond du plat. La lamelle va tomber en se déplaçant du côté de glisser sur le côté. </li><li> Après la lamelle est retirée, placer la lame dans un rack d&#39;un bain chaud frais avec 1XSSCm SDS à 0,05%. Agiter pendant quelques minutes </li><li> Transfert diapositives plus porte à un bain de 0,06 X SSC. </li><li> Transfert diapositives à l&#39;autre bae de 0,06 X SSC contenant un rack frais, buvard la lame sur une serviette en papier pour enlever autant de SDS-tampon contenant que possible avant de placer dans le bain frais. </li><li> Agiter diapositives pour un couple de minutes. </li><li> Spin immédiatement à la température ambiante pendant 5 min à 700 rpm, puis glisse êtes prêt à numériser. Conserver dans l&#39;obscurité jusqu&#39;à ce numérisées. </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

Analyse bactérienne expression génique par puces à ADN

bacterial-gene-expression-analysis-using-microarrays

Research

JoVE Journal

Biology

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

Vidéo: 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 préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

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

Recherche

Biologie

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.

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

Infection virus de la vaccine et l&#39;analyse temporelle de l&#39;expression des gènes de virus: Partie 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 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.

Infection virus de la vaccine et l&#39;analyse temporelle de l&#39;expression des gènes de virus: Partie 3

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.

Mondial Analyse expression génique utilisant une plateforme biopuces oligonucléotidiques Zebrafish

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

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Spinal Cord électrophysiologie

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

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.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

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.

Aide d&#39;un compteur de cellules automatisé pour simplifier les études d&#39;expression génique: Knockdown siRNA de l&#39;IL-4 dans l&#39;expression des gènes dépendant cellules 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.

Analyse de l&#39;expression génique dans l&#39;agrile du frêne ( Agrilus planipennis) En utilisant quantitative en temps réel-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 ...

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.

L&#39;échelle du génome Analyse des aminoacylation (recharge) Niveaux d&#39;ARNt utilisant des 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 ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Les articles inclus en paraffine et congelés de Drosophile</emMuscles adultes>

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...