Reação reversa Transcriptase Vire bloco de aquecimento a 70-80 ° C e 42 ° C. Banhos de água também pode ser usado nesta etapa. Se estiver usando blocos de aquecimento, coloque um pouco de água para que o calor é uniforme. <ol><li> Reação priming <ol><li> Tome 3 mg de RNA </li><li> Ajustar o volume para 13 mL com água livre de RNase </li><li> Adicionar 2,5 mL de aleatório hexâmero primers (2mg/ml) </li></ol></li><li> Misturar bem e incubar a 70-80 ° C por 8 minutos. Em seguida, coloque, em gelo por 5 min. </li><li> Prepare RT cocktail (14,5 mL / rxn): <table border="0"><tbody><tr><td> Componente </td><td> Volume </td></tr><tr><td> Tampão 5X First Strand </td><td> 6 mL </td></tr><tr><td> 0,1 M DTT </td><td> 3 mL </td></tr><tr><td> Mix dNTP aminoallyl-25X </td><td> 1,2 mL </td></tr><tr><td> SuperScript II RT (200U/μl) </td><td> 2,3 mL </td></tr><tr><td> Água </td><td> 2,0 mL </td></tr></tbody></table> * Para as reacções n, prepare Mix Master de n 0,5 alíquotas de cocktail para garantir que você não vai acabar. </li><li> Adicionar 14,5 mL de reações resfriado. </li><li> Mix (não vortex) e incubar a 42 ° C por 3-4 horas </li><li> Amostras podem ser armazenadas a -20 ° C, se necessário. </li></ol> Hidrolisar RNA <ol><li> Para cada amostra, adicionar 3 mL de NaOH 1 N e 0,6 mL de 0,5 M EDTA. (Final conc.s = NaOH 100mM, EDTA 10mM) </li><li> Misturar e incubar a 65 ° C por 10 minutos. </li><li> Neutralize adicionando 33,6 mL HEPES 1M, pH = 7,0 (final conc .= 500mM HEPES) </li><li> Amostras podem ser armazenadas a -20 ° C, se necessário. </li></ol> Purificação de reação: Remoção de unincorporated aa-dUTP e aminas livres Nota: Este protocolo de purificação é modificado a partir da PCRpurification QIAquick Qiagen protocolo kit. A lavagem de fosfato e buffers de eluição são substituídos pela Qiagen buffers fornecidos porque os buffers Qiagen contêm aminas livres que competem com a reação de acoplamento Cy corante. As colunas do kit de mini-prep também pode ser usado. <ol><li> Mistura de reacção cDNA com 300 ml (ou volume de reação 5X = mL 336) tampão PB (Qiagen fornecido) e transferir para a coluna QIAquick. </li><li> Coloque a coluna em um tubo de coleta de 2 ml (Qiagen fornecido) e centrifugar a 13.000 rpm ~ por 1 minuto. Tubo de coleta vazio. </li><li> Para lavar, adicionar 750 mL de fosfato tampão de lavagem (não Qiagen) para a coluna e centrifugar a 13.000 rpm ~ por 1 minuto. </li><li> Esvaziar o tubo de coleta e repetir a lavagem 750 mL e etapa de centrifugação. </li><li> Esvaziar o tubo de coleta e centrifugar a coluna um adicional de 1 minuto na velocidade máxima. </li><li> Transferência da coluna para um novo microtubo de 1,5 mL e com cuidado, 30 mL de fosfato tampão de eluição. (Ver secção preparação da solução) </li><li> Incubar por 1 minuto à temperatura ambiente. </li><li> Eluir por centrifugação a 13.000 rpm ~ por 1 minuto. </li><li> Eluir uma segunda vez (com outra mL 30 de tampão de eluição fosfato) no mesmo tubo, repetindo as etapas 6-8. O volume de eluição final deve ser ~ 60 mL. </li><li> A quantidade de cDNA podem ser quantificados nesta fase: ng cDNA = (fator de diluição) (OD260) (37) (volume total eluído da coluna) </li><li> Amostra seca em uma velocidade vac a 45 ° C. </li><li> Amostras podem ser armazenadas a -20 ° C, se necessário. </li></ol> Acoplamento aa-cDNA para Cy Ester Dye <ol><li> Ressuspender aminoallyl marcado cDNA com 10 ml 50 mM bicarbonato de sódio, pH = 9,0, se necessário. (Esta reserva deve ser feita nova a cada duas semanas) </li><li> Trabalhando em um lugar escuro, adicione a sonda a um tubo do corante Cy apropriado. Pipeta para cima e para baixo várias vezes para voltar a suspender o corante. </li><li> Incubar a reação de uma hora no escuro à temperatura ambiente. (Incubação pode ir até duas horas) </li></ol> Purificação reação II: Remoção de corante desacoplado <ol><li> À reação 35 mL adicionar 100 mM pH 5,2 NaOAc. Misture bem. </li><li> Adicionar 250 mL (ou volume de reação 5X = 225 mL) Buffer PB (Qiagen fornecido) para cada amostra e misture pipetando para cima e para baixo. </li><li> Coloque a coluna QIAquick em um tubo de coleta de 2 mL (Qiagen fornecido), aplicam-se a amostra para a coluna, e centrifugar a ~ 13.000 para 1 minuto. Tubo de coleta vazio. </li><li> Para lavar, adicionar 0,75 mL de tampão PE (Qiagen fornecido) para a coluna e centrifugar a ~ 13.000 para 1 minuto. </li><li> Repita o passo 4. </li><li> Tubo de coleta vazio e coluna centrífuga para um 1 minuto adicional na velocidade máxima. </li><li> Coloque em uma coluna limpa microtubo de 1,5 mL e adicionar 30 mL cuidadosamente tampão EB (Qiagen fornecido) para o centro da membrana da coluna. </li><li> Incubar por 1 minuto à temperatura ambiente. </li><li> Eluir por centrifugação a 13.000 rpm ~ por 1 minuto. </li><li> Eluir pela segunda vez no mesmo tubo, repetindopassos 7-9. O volume final deve ser eluída ~ mL 60. </li></ol> Análise da reação de Rotulagem A quantidade de cDNA e etiqueta pode ser quantificado nesta fase: <ul><li> Medida OD 260, 550 e 650 (em branco é a água.) Use o programa microarray nanodrop. </li><li> Calcular o pmol de corantes incorporação e DNA pmol e nuc / corante relação com as equações abaixo: nucleotídeos pmol = [OD260 * volume (mL) * 37 ng / mL * 1000 pg / ng] / 324,5 pg / pmol </li></ul> Nota: 1 OD260 = 37 ng / mL para cDNA; 324,5 pg / pmol peso molecular médio de um dNTP) pmol Cy3 OD550 = volume * (mL) / 0,15 pmol Cy5 = OD650 * volume (mL) / 0,25 nucleotídeos / relação dye = cDNA pmol / pmol Cy dye Se você estiver usando nanodrop, já dá pmol / mL para cada corante. Você só precisa multiplicar esse número por volume para obter a incorporação de corantes totais pmol. Nota:&gt; 150-200 pmol de incorporação de corante por amostra e uma proporção de menos de 20 nucleotídeos / moléculas de corante é ideal para hibridizações. <ul><li> Amostras podem ser armazenadas a -20 ° C no escuro. <ul><li> Combine sondas a ser hibridizado juntos e seco no speedvac a 42 ° C. </li><li> Amostras podem ser armazenadas a -20 ° C até estar pronto para hyb. </li></ul></li></ul> Pré-hyb slides <ol><li> Slides lugar rosto para baixo sobre RT 100 mL de água em um suporte de lâmina limpa vermelha por 3-5 minutos </li><li> Encaixe a lâmina seca, colocando a face para cima em um bloco de aquecimento 100 ° C por alguns segundos - relógio para a condensação de desaparecer </li><li> UV ligação transversal a lâmina de 250 mJoules (Digite 2500 para o reticulador UV) </li><li> Slides hidratar por imersão em pré-aquecido a água 42 C °. Spin-5 min a 700 RPM na RT. </li><li> Slides incubar em jarra de Coplin 50ml contendo pré-aquecido pré-hyb (5XSSC, BSA 1%, 0,1% SDS) para pelo menos 45 minutos @ 42 ° C, e de incubação pode ir mais longe, se necessário. 50 buffer de pré-hyb mL: 12,5 mL 20x SSC, 10 ml BSA 5%, 0,5 ml SDS 10%, 27 mL de água </li><li> Dip slides na pré-aquecido a água (42 ° C) e agitar um par de minutos para remover o pré-hyb buffer. </li><li> Enxágüe em isopropanol e spin 5 min a 700 rpm, temperatura ambiente, para secar. </li></ol> Amostras preparando para a hibridização <ol><li> Ressuspender sonda em água (volumes são apresentados abaixo) <table border="0"><tbody><tr><td> Para vol total. = 30μl </td><td> Para vol total. = 35μl </td><td> Para o total vol .= 40μl </td><td></td></tr><tr><td> 19,8 mL de água </td><td> 23,55 mL de água </td><td> 27,2 mL de água </td><td> </td></tr><tr><td> 1,5 mL </td><td> 1,5 mL </td><td> 1,5 mL </td><td> 10 mg / ml de DNA de esperma de salmão (Invitrogen) </td></tr><tr><td> 1,2 mL </td><td> 1,2 mL </td><td> 1,2 mL </td><td> 12,5 mg / ml tRNA </td></tr><tr><td> 4,5 mL </td><td> 5,25 mL </td><td> 6 mL </td><td> 20XSSC (final 3x) </td></tr><tr><td> 3 mL </td><td> 3,5 mL </td><td> 4 mL </td><td> 1% SDS </td></tr></tbody></table> Nota: A ordem de adição dos reagentes são importantes. Não preparar mix master. </li><li> Ferva por 5 minutos, depois de spin 5 minutos a 14.000 rpm. Esfriar por 2 min à temperatura ambiente. </li></ol> Hibridização <ol><li> Adicionar 10-12 mL de 3X SSC aos poços da câmara de hibridação em cada ponta e no meio. </li><li> Adicionar um deslizamento levantador limpa para a área apropriada da lâmina e gentilmente carga em solução de hibridização (30 mL -40). Deve haver em cada amostra adicional borda da lamínula. </li><li> Coloque o slide na câmara. Aperte os parafusos e colocar em banho-maria a 65 ° C durante a noite. Não coloque a câmara diretamente sobre o fundo do banho-maria. Coloque um bloco no topo da câmara superior para manter tudo no lugar. Mantenha a tampa do banho de água para proteger as amostras sensíveis à luz. </li></ol> Hyb Lava <ol><li> Preparar os buffers seguinte: <ul><li> SSC 1X, SDS 0,05% </li><li> 0,06 X SSC </li></ul></li><li> Depois de a câmara de incubação de hibridação unseal. Remove o slide da câmara, tomando cuidado para não perturbar a lamela. </li><li> Para remover lamelas, lâminas de submergir em um prato quente contendo 1X SSC, 0,05% SDS lamela tampão de lavagem de frente para o fundo do prato. A lamela cairá movendo o lado deslize para o lado. </li><li> Após a lamínula é retirada, coloque o slide em um rack de um banho quente fresco com 1XSSCm SDS 0,05%. Agitar por alguns minutos </li><li> Transferência de slides mais rack para um banho de 0,06 X SSC. </li><li> Transferência de slides para outro baª de 0,06 X SSC contendo um rack novo, apagando o slide sobre um papel toalha para remover o máximo tampão contendo SDS-quanto possível antes de colocar no banho fresco. </li><li> Agitar slides para um par de minutos. </li><li> Girar imediatamente à temperatura ambiente por 5 min @ 700 rpm, em seguida, slides estão prontos para fazer a varredura. Loja no escuro até digitalizada. </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>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<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

Análise de Expressão Gênica Utilizando bacteriana Microarrays

bacterial-gene-expression-analysis-using-microarrays

Research

JoVE Journal

Biology

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

Vídeo: 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.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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

Biologia

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.

Infecção pelo Vírus Vaccinia e Análise Temporal da Expressão Gênica de Vírus: 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 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.

Infecção pelo Vírus Vaccinia e Análise Temporal da Expressão Gênica de Vírus: Parte 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.

Análise da expressão global de Gene usando uma plataforma Microarray Zebrafish Oligonucleotide

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 Eletrofisiologia

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.

Usando o Sistema de Eletroporação Gene Pulser MXcell para transfectar pilhas com Alta Eficiência

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.

Usando um contador de células automatizadas para simplificar Estudos da Expressão Gênica: Knockdown siRNA de Expressão IL-4 Gene Dependente em células 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.

Análise de Expressão Gênica em Emerald Borer Ash ( Agrilus planipennis) Usando Quantitativa em Tempo Real-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.

Análise do genoma de Aminoacylation (carregamento) Níveis de tRNA Usando 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.

Seções incluído em parafina e congelados de Drosophila Músculos Adulto

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