Whitehead Institute Fellows Fundos

Parte 1: síntese de cDNA a partir de RNA <ol><li> Antes de usar o Ambion Amino Allyl MessageAmp II kit, adicionar o volume recomendado de etanol a 100% para os buffers de lavagem. </li><li> PCR em tubo de reação, adicione entre 100ng de 5μg de RNA total e 1μl de T7 oligo primer (dT). Trazer o volume até 12μl com água livre de nuclease. </li><li> Incubar as amostras a 70 ° C por 10 min em um termociclador. </li><li> Remover amostras de RNA a partir de 70 ° C e centrifugar brevemente. Colocar no gelo. </li><li> Preparar o 1 º Strand master mix Síntese e manter a temperatura ambiente (com excesso de 10% para pipetagem de erro). (Tabela 1) </li><li> Pipeta suavemente Mix Master ou filme para misturar e então centrifugar brevemente. </li><li> Transferência 8μl do Mix Master para cada amostra de RNA. Mix pipetando cima e para baixo 3-4 vezes. </li><li> Incubar a 42 ° C por 2 horas no termociclador. </li><li> Brevemente centrifugar amostras e colocar no gelo. Prosseguir imediatamente para a próxima etapa de síntese de dsDNA. </li><li> Prepare o 2 º Strand master mix de síntese sobre gelo (com sobrecarga de 10% para pipetagem de erro). (Tabela 2) </li><li> Pipeta suavemente Mix Master ou filme para misturar e então centrifugar brevemente. </li><li> Transferência Pipete 80 para cada amostra e misture suavemente pipetando cima e para baixo 3-4 vezes. </li><li> Incubar a 16 ° C por 2 horas no termociclador. </li><li> Após a incubação de 2 horas, prosseguir com a etapa de clean-up cDNA ou congelar imediatamente a -20 ° C. </li></ol> Parte 2: Double-stranded cDNA clean-up <ol><li> Remover o Pure cDNA do frigorífico e deixe-o atingir a temperatura ambiente por 30 minutos antes de usar. Agitar o frasco plenamente ressuspender o cDNA magnética contas obrigatórias antes do uso. </li><li> Alíquota de água livre de nuclease em um tubo de 1,5 ml e incubar a 50-60 ° C durante pelo menos 10 minutos durante a incubação prévia 2hr. </li><li> Adicionar 180μl de Pure cDNA de cada amostra, e misturar completamente por pipetagem cima e para baixo. Transferir as amostras para uma placa de 96 poços de fundo redondo. </li><li> Continue a misturar as amostras agitando a placa em agitador orbital por pelo menos 2 minutos. </li><li> Mova a placa para um suporte magnético para capturar a esferas magnéticas. Deixe placa no stand por aproximadamente 6 minutos, ou até que a mistura torna-se transparente e os cordões de ligação a peletizada. </li><li> Aspirar cuidadosamente o sobrenadante com um aspirador a vácuo, sem perturbar a esferas magnéticas. Como alternativa, remova cuidadosamente o sobrenadante com uma pipeta e desprezar o sobrenadante. </li><li> Retire a placa do suporte magnético. </li><li> Adicionar 150μl tampão de lavagem cDNA a cada poço e agitar a placa por 1 minuto no agitador orbital a uma velocidade moderada. Contas não se dispersa nesta etapa, devido à baixa tensão superficial da solução de lavagem. </li><li> Mova a placa para um suporte magnético para capturar a esferas magnéticas. </li><li> Aspirar cuidadosamente o sobrenadante com um aspirador a vácuo, sem perturbar a esferas magnéticas. Como alternativa, remova cuidadosamente o sobrenadante com uma pipeta e desprezar o sobrenadante. </li><li> Retire a placa do suporte magnético. </li><li> Repita a lavagem de cada vez 2 º com tampão de lavagem 150μl cDNA. </li><li> Após a lavagem 2 º, seca as contas agitando a placa por 2 minutos no agitador orbital à velocidade máxima. Não overdry! </li><li> Eluir o cDNA dos grânulos adicionando 18μl da água livre de nuclease pré-aquecido a cada amostra. </li><li> Agitar vigorosamente a placa por 3 minutos no agitador orbital, em seguida, verifique se a esferas magnéticas estão totalmente dispersos. Se não, continue a tremer. </li><li> Uma vez que a esferas magnéticas têm totalmente disperso, mova a placa a um suporte magnético para capturar as esferas magnéticas. </li><li> Transferir cuidadosamente o cDNA eluída (~ 16μl) para uma placa de PCR nova (ou tubos de PCR). </li><li> Prosseguir diretamente para a etapa seguinte, ou congelar o cDNA a -20 ° C. </li></ol> Parte 3: transcrição in vitro (IVT) <ol><li> Prepare a mistura mestre IVT à temperatura ambiente. (Tabela 3) </li><li> Pipeta suavemente o Mix Master ou filme para misturar e centrifugar brevemente. </li><li> Adicionar 24μl de cada amostra, e misture delicadamente por pipetagem cima e para baixo 3-4 vezes. </li><li> Incubar a 37 ° C por 14 horas em um termociclador, então segure a 4 ° C até que esteja pronto para a próxima etapa. </li></ol> Parte 4: ARNA clean-up após IVT <ol><li> Vortex as contas RNA ligação rapidamente para obter uma mistura mesmo antes do uso. </li><li> Preparar a mistura ARNA Binding à temperatura ambiente (Tabela 4) Isso pode ser feito antes do tempo -. A mistura preparada ligação pode ser armazenado em temperatura ambiente por até uma semana. </li><li> Misture bem em vórtice. </li><li> Alíquota do tampão de eluição ARNA em um tubo de 1,5 ml e incubar a 50-60 ° C durante pelo menos 10 minutos. </li><li> Adicionar 70μl da mistura obrigatória ARNApara cada amostra e misture bem por pipetagem cima e para baixo 3-4 vezes. </li><li> Transferência das amostras da placa de PCR para uma placa de 96 poços de fundo redondo. </li><li> Adicionar 50μl de isopropanol 100% a cada amostra e misture bem por pipetagem cima e para baixo 3-4 vezes. </li><li> Agitar suavemente a placa em agitador orbital por pelo menos 2 minutos para misturar as amostras. </li><li> Mova a placa para um suporte magnético para capturar a esferas magnéticas. Deixar a placa no stand até que a mistura torna-se transparente e os cordões de ligação a peletizada. </li><li> Aspirar cuidadosamente o sobrenadante com um aspirador a vácuo, sem perturbar a esferas magnéticas. Como alternativa, remova cuidadosamente o sobrenadante com uma pipeta e desprezar o sobrenadante. </li><li> Retire a placa do suporte magnético. </li><li> Adicionar 100μl de solução de lavagem ARNA a cada poço e agitar a placa por 1 minuto no agitador orbital a uma velocidade moderada. Contas não pode se dispersar totalmente nesta etapa. </li><li> Mova a placa para um suporte magnético para capturar a esferas magnéticas. </li><li> Aspirar cuidadosamente o sobrenadante com um aspirador a vácuo, sem perturbar a esferas magnéticas. Como alternativa, remova cuidadosamente o sobrenadante com uma pipeta e desprezar o sobrenadante. </li><li> Retire a placa do suporte magnético. </li><li> Repita a lavagem de cada vez 2 º com 100μl de solução de lavagem ARNA. </li><li> Após a lavagem 2 º, seca as contas agitando a placa por 1 minuto no agitador orbital à velocidade máxima. Não overdry as amostras! </li><li> Eluir a ARNA dos grânulos adicionando 40μl tampão de eluição ARNA pré-aquecido para cada amostra. </li><li> Agitar vigorosamente a placa no agitador orbital por 3 minutos, em seguida, verifique se a esferas magnéticas estão totalmente dispersos. Se não, continue a tremer. </li><li> Uma vez que a esferas magnéticas têm totalmente disperso, mova a placa a um suporte magnético para capturar as esferas magnéticas. O sobrenadante contém a limpo-up amino-alil ARNA incorporadas. </li><li> Transferir cuidadosamente o ARNA eluída a uma placa de PCR nova (ou tubos de PCR). </li><li> (Passo opcional) Verificar a concentração de RNA das amostras medindo 1.5μl em um espectrofotômetro NanoDrop. </li><li> Imediatamente continue para a próxima etapa, ou armazenar a ARNA a -80 ° C. </li></ol> Tabela 1: 1 Strand cDNA º Mix Master Síntese <table border="1" cellpadding="8"><tbody><tr><td> Reagente </td><td> Montante para uma reação </td></tr><tr><td> Tampão Strand 10X Primeiro </td><td> 2 l </td></tr><tr><td> dNTP Mix </td><td> 4 mL </td></tr><tr><td> Inibidor ribonuclease </td><td> 1 ml </td></tr><tr><td> Script da Matriz </td><td> 1 ml </td></tr></tbody></table> Tabela 2: 2 ª Strand cDNA Mix Master Síntese <table border="1" cellpadding="8"><tbody><tr><td> Reagente </td><td> Montante para uma reação </td></tr><tr><td> Água livre de nuclease </td><td> 63 mL </td></tr><tr><td> Tampão 10X Segundo Strand </td><td> 10 ml </td></tr><tr><td> dNTP Mix </td><td> 4 mL </td></tr><tr><td> DNA Polimerase </td><td> 2 l </td></tr><tr><td> RNase H </td><td> 1 ml </td></tr></tbody></table> Tabela 3: IVT Master Mix <table border="1" cellpadding="8"><tbody><tr><td> Reagente </td><td> Montante para uma reação </td></tr><tr><td> aaUTP solução (75 mM) </td><td> 2 l </td></tr><tr><td> ATP, CTP, GTP mix (25 mM) </td><td> 12 mL </td></tr><tr><td> T7 UTP solução (75 mM) </td><td> 2 l </td></tr><tr><td> T7 Tampão de reacção 10X </td><td> 4 mL </td></tr><tr><td> T7 Mix Enzyme </td><td> 4 mL </td></tr></tbody></table> Tabela 4: ARNA Mix Binding <table border="1" cellpadding="8"><tbody><tr><td> Reagente </td><td> Montante para uma reação </td></tr><tr><td> Beads RNA Binding * </td><td> 10 ml </td></tr><tr><td> Bead * Solução ressuspensão </td><td> 4 mL </td></tr><tr><td> Isopropanol 100% ** </td><td> 6 mL </td></tr><tr><td> ARNA Concentrado de tampão de ligação </td><td> 50 ul </td></tr></tbody></table> * Mix as contas RNA ligação com a solução de ressuspensão primeira conta. ** Adicione o isopropanol e misturar bem antes de adicionar o ARNA concentrado de tampão de ligação.

<ol>
	<li>Rubins, K. H., Hensley, L. E., Bell, G. W., Wang, C., Lefkowitz, E. J., Brown, P. O., Relman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+viral+gene+expression+programs+during+poxvirus+infection:+a+transcriptional+map+of+the+vaccinia+and+monkeypox+genomes.">Comparative analysis of viral gene expression programs during poxvirus infection: a transcriptional map of the vaccinia and monkeypox genomes.</a> PLoS ONE. 3 (7), e2628-e2628 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+a+complete+poxvirus+transcriptome+reveals+an+immediate-early+class+of+genes.">Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes.</a> Proc. Natl. Acad. Sci. 105 (6), 2140-2145 (2008).</li><li>Satheshkumar, P. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poxvirus+transcriptome+analysis.">Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E62-E62 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A.+Reply+to+Satheshkumar+and+Moss:+Poxvirus+transcriptome+analysis.">A. Reply to Satheshkumar and Moss: Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E63-E64 (2008).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., Mu&#241;oz, M., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+gene+expression+survey+of+vaccinia+virus+infection+of+human+HeLa+cells.">Cellular gene expression survey of vaccinia virus infection of human HeLa cells.</a> J Virol. 77 (11), 6493-6506 (2003).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Conde, R., Pascual-Montano, A., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+reveals+characteristic+changes+of+host+cell+gene+expression+in+response+to+attenuated+modified+vaccinia+virus+Ankara+infection+of+human+HeLa+cells.">Microarray analysis reveals characteristic changes of host cell gene expression in response to attenuated modified vaccinia virus Ankara infection of human HeLa cells.</a> J Virol. 78 (11), 5820-5824 (2004).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., N&#225;jera, J. L., Zaballos, A., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+response+to+the+attenuated+poxvirus+vector+NYVAC:+upregulation+of+apoptotic+genes+and+NF-kappaB-responsive+genes+in+infected+HeLa+cells.">Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.</a> J Virol. 80 (2), 985-998 (2006).</li><li>Guerra, S., N&#225;jera, J. L., Gonz&#225;lez, J. M., L&#243;pez-Fern&#225;ndez, L. A., Climent, N., Gatell, J. M., Gallart, T., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+gene+expression+profiling+after+infection+of+immature+human+monocyte-derived+dendritic+cells+by+the+attenuated+poxvirus+vectors+MVA+and+NYVAC.">Distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors MVA and NYVAC.</a> 81 (16), 8707-8721 (2007).</li></ol>

The authors have nothing to disclose.

Passos crítica A segunda etapa de amplificação não é aconselhável como preconceitos em dados de matriz foram observados. Misturando com cuidado a cada passo enzimático (1 ª e 2 ª vertente de síntese de cDNA, FPI) é fundamental para a obtenção de rendimentos de amplificação boa, como é de incubação de cada etapa enzimática na temperatura adequada. A cycler PCR com tampa aquecida ajustável é o preferido - desvios ainda tão pequeno quanto 2-3 graus dura...

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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>Amino Allyl MessageAmp II aRNA amplification kit</td>
<td>Reagent</td>
<td>Applied Biosystems</td>
<td>AM1753</td>
<td>For 20 reactions</td>
</tr>
<tr>
<td>Amino Allyl MessageAmp II aRNA amplification kit</td>
<td>Reagent</td>
<td>Applied Biosystems</td>
<td>AM1821</td>
<td>For 100 reactions, in a 96-well format</td>
</tr>
<tr>
<td>NanoDrop ND-1000 UV-VIS spectrophotometer</td>
<td>Other</td>
<td>NanoDrop</td>
<td>ND-1000</td>
<td>Or equivalent spectrophotometer</td>
</tr>		</tbody>
		</table>
		</div>

vaccinia virus infection &amp; temporal analysis of virus gene expression: part 2

A família<em&gt; Poxviridae</em&gt; Consiste de DNA double-stranded grandes contendo vírus que se replicam exclusivamente no citoplasma das células infectadas. Membros da<em&gt; Orthopox</em&gt; Gênero incluem a varíola, o agente causador da varíola humana, macacos, e vaccinia (VAC), o membro protótipo da família de vírus. Dentro do relativamente grande (~ 200 kb) vaccinia genoma, três classes de genes são codificados: precoce, intermediária e final. Enquanto todas as três classes são transcritos pela polimerase viral codificado RNA, cada classe tem uma função diferente no ciclo de vida do vírus. Poxvírus utilizar múltiplas estratégias para a modulação do ambiente celular do hospedeiro durante a infecção. A fim de compreender a regulação de ambos os host e expressão do gene do vírus, nós utilizamos genome-wide abordagens para analisar a abundância transcrição de ambos os vírus e células hospedeiras. Aqui, demonstramos infecções tempo de percurso de células HeLa com Vaccinia vírus RNA e amostragem em vários intervalos de tempo pós-infecção. Ambos os host e viral RNA total é isolado e amplificado para a hibridização de microarrays para a análise da expressão gênica.

Watch this Scientific Journal Video about Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2 at JoVE.com

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

Protocolo para Vaccinia infecção de células HeLa e análise de host e expressão gênica viral. Parte 2 de 3.

Infecção pelo Vírus Vaccinia e Análise Temporal da Expressão Gênica de Vírus: Parte 2

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-temporal-analysis-virus-gene-expression-part

Research

JoVE Journal

Biology

7.9K visualizações.  MIT - Massachusetts Institute of Technology. Protocolo para Vaccinia infecção de células HeLa e análise de host e expressão gênica viral. Parte 2 de 3.

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

Bacterial Gene Expression Analysis Using Microarrays

Análise de Expressão Gênica Utilizando bacteriana Microarrays

Biologia

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 ...

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

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

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Induzida por vírus silenciamento gênico (VIGS) em Nicotiana benthamiana Tomate e

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

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 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Agrobacterium-Mediated Virus-Induced Assay silenciamento gênico Em Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

A Plataforma de Produção de Proteína Complexo MultiBac no EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Análise da Global RNA Síntese no nível da célula única seguinte hipóxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...