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Parte 1: sintesi di cDNA da RNA <ol><li> Prima di utilizzare il Amino Ambion allile MessageAmp II kit, aggiungere il volume consigliato del 100% di etanolo al tamponi di lavaggio. </li><li> In tubo di reazione PCR, aggiungere tra 5μg a 100 ng di RNA totale e 1ml di T7 oligo (dT) primer. Portare il volume fino a 12μl con acqua priva di nucleasi. </li><li> Incubare i campioni a 70 ° C per 10 minuti in un termociclatore. </li><li> Rimuovere campioni di RNA da 70 ° C e centrifugare brevemente. Mettere in ghiaccio. </li><li> Preparare lo Strand 1 ° mix di sintesi master e tenere a temperatura ambiente (con il 10% carico eccessivo per il pipettaggio errore). (Tabella 1) </li><li> Delicatamente Master Mix pipetta o sfiorare per mescolare, quindi si centrifuga brevemente. </li><li> Trasferimento 8μl del Master Mix per ogni campione di RNA. Mescolare pipettando su e giù per 3-4 volte. </li><li> Incubare a 42 ° C per 2 ore in termociclatore. </li><li> Centrifugare i campioni brevemente e posto sul ghiaccio. Procedere immediatamente alla fase successiva della sintesi dsDNA. </li><li> Preparare il Settore 2 ° mix di sintesi master sul ghiaccio (con il 10% carico eccessivo per il pipettaggio errore). (Tabella 2) </li><li> Delicatamente Master Mix pipetta o sfiorare per mescolare, quindi si centrifuga brevemente. </li><li> Trasferire 80μl di ciascun campione e mescolare delicatamente pipettando su e giù per 3-4 volte. </li><li> Incubare a 16 ° C per 2 ore in termociclatore. </li><li> Dopo l&#39;incubazione 2 ore, procedere con il cDNA clean-up o step congelare immediatamente a -20 ° C. </li></ol> Parte 2: doppia elica del DNA di pulizia <ol><li> Rimuovere il puro cDNA dal frigorifero e lasciarlo equilibrare a temperatura ambiente per 30 minuti prima dell&#39;uso. Agitare il flacone per sospendere di nuovo pienamente le sfere magnetiche cDNA vincolante prima dell&#39;uso. </li><li> Aliquota nucleasi senza acqua in un tubo di 1,5 ml ed incubare a 50-60 ° C per almeno 10 minuti durante l&#39;incubazione precedente 2 ore. </li><li> Aggiungere 180μl di Pure cDNA per ogni campione, e mescolare bene pipettando su e giù. Trasferire i campioni ad un 96-ben a fondo rotondo piatto. </li><li> Continuare a mescolare i campioni scuotendo leggermente il piatto su un agitatore orbitale per almeno 2 minuti. </li><li> Spostare la piastra ad un supporto magnetico per catturare le sfere magnetiche. Lascia piastra sul supporto per circa 6 minuti, o fino a quando il composto diventa trasparente e le palline sono vincolanti pellet. </li><li> Con attenzione aspirare il surnatante con un aspiratore a vuoto senza disturbare il sfere magnetiche. In alternativa, rimuovere con attenzione il surnatante con una pipetta e scartare il surnatante. </li><li> Togliere la placca dal supporto magnetico. </li><li> Aggiungere 150μl cDNA tampone di lavaggio in ogni bene e agitare la piastra per 1 minuto l&#39;agitatore orbitale a velocità moderata. Perline NON si disperderà in questa fase, a causa della bassa tensione superficiale del tampone di lavaggio. </li><li> Spostare la piastra ad un supporto magnetico per catturare le sfere magnetiche. </li><li> Con attenzione aspirare il surnatante con un aspiratore a vuoto senza disturbare il sfere magnetiche. In alternativa, rimuovere con attenzione il surnatante con una pipetta e scartare il surnatante. </li><li> Togliere la placca dal supporto magnetico. </li><li> Ripetere il lavaggio per volta 2 ° con 150μl tampone di lavaggio cDNA. </li><li> Dopo il 2 ° lavare, asciugare le perle agitando la piastra per 2 minuti in agitatore orbitale alla massima velocità. Non asciugare eccessivamente! </li><li> Eluire il cDNA dalla perle aggiungendo 18μl del preriscaldato nucleasi acqua priva di ogni campione. </li><li> Agitare vigorosamente la piastra per 3 minuti mediante l&#39;agitatore orbitale, quindi controllare per assicurarsi che le sfere magnetiche sono completamente disperse. In caso contrario, continuare a tremare. </li><li> Una volta che il sfere magnetiche sono completamente dispersi, spostare la piastra ad un supporto magnetico per catturare le sfere magnetiche. </li><li> Trasferire accuratamente l&#39;cDNA eluiti (~ 16μl) ad una nuova piastra PCR (PCR o tubi). </li><li> Procedere direttamente alla fase successiva, o congelare il cDNA a -20 ° C. </li></ol> Parte 3: Trascrizione in vitro (IVT) <ol><li> Preparare la master mix IVT a temperatura ambiente. (Tabella 3) </li><li> Delicatamente pipetta Master Mix o sfiorare per miscelare e centrifugare brevemente. </li><li> Aggiungere 24μl di ciascun campione e mescolare delicatamente pipettando su e giù per 3-4 volte. </li><li> Incubare a 37 ° C per 14 ore in un termociclatore, quindi tenere a 4 ° C fino al momento per il passo successivo. </li></ol> Parte 4: ARNA pulizia dopo IVT <ol><li> Vortex le perline RNA vincolante per breve tempo ad ottenere un impasto omogeneo prima dell&#39;uso. </li><li> Preparare il Mix Binding ARNA a temperatura ambiente (Tabella 4) Questo può essere fatto prima del tempo -. Mix preparato vincolante può essere conservato a temperatura ambiente per un massimo di una settimana. </li><li> Mescolare bene nel vortex. </li><li> Aliquota del buffer di eluizione ARNA in un tubo di 1,5 ml ed incubare a 50-60 ° C per almeno 10 minuti. </li><li> Aggiungere 70μl della miscela ARNA vincolantead ogni campione e mescolare bene pipettando su e giù per 3-4 volte. </li><li> Trasferimento dei campioni dalla piastra PCR ad un 96-ben a fondo rotondo piatto. </li><li> Aggiungere 50μl del 100% di isopropanolo a ciascun campione e mescolare bene pipettando su e giù per 3-4 volte. </li><li> Agitare delicatamente la piastra su un agitatore orbitale per almeno 2 minuti per mescolare completamente i campioni. </li><li> Spostare la piastra ad un supporto magnetico per catturare le sfere magnetiche. Lascia la piastra sul supporto fino a quando il composto diventa trasparente e le palline sono vincolanti pellet. </li><li> Con attenzione aspirare il surnatante con un aspiratore a vuoto senza disturbare il sfere magnetiche. In alternativa, rimuovere con attenzione il surnatante con una pipetta e scartare il surnatante. </li><li> Togliere la placca dal supporto magnetico. </li><li> Aggiungere 100μl soluzione di lavaggio ARNA in ogni pozzetto e agitare la piastra per 1 minuto l&#39;agitatore orbitale a velocità moderata. Perline potrebbero non disperdere in questa fase. </li><li> Spostare la piastra ad un supporto magnetico per catturare le sfere magnetiche. </li><li> Con attenzione aspirare il surnatante con un aspiratore a vuoto senza disturbare il sfere magnetiche. In alternativa, rimuovere con attenzione il surnatante con una pipetta e scartare il surnatante. </li><li> Togliere la placca dal supporto magnetico. </li><li> Ripetere il lavaggio per volta 2 ° con la soluzione di 100μl Wash ARNA. </li><li> Dopo il 2 ° lavare, asciugare le perle agitando la piastra per 1 minuto l&#39;agitatore orbitale alla massima velocità. Non asciugare eccessivamente i campioni! </li><li> Eluire la ARNA dalle perle con l&#39;aggiunta di 40μl preriscaldato Elution Buffer ARNA per ogni campione. </li><li> Agitare vigorosamente la piastra l&#39;agitatore orbitale per 3 minuti, quindi controllare per assicurarsi che le sfere magnetiche sono completamente disperse. In caso contrario, continuare a tremare. </li><li> Una volta che il sfere magnetiche sono completamente dispersi, spostare la piastra ad un supporto magnetico per catturare le sfere magnetiche. Il supernatante contiene la ARNA ripulito amino-allil incorporato. </li><li> Trasferire accuratamente l&#39;ARNA eluiti ad una nuova piastra PCR (PCR o tubi). </li><li> (Passo opzionale) Controllare la concentrazione di RNA dei campioni misurando 1.5μl su uno spettrofotometro NanoDrop. </li><li> Immediatamente continuare al passaggio successivo, o lasciare il ARNA a -80 ° C. </li></ol> Tabella 1: Mix Strand cDNA 1 ° master di sintesi <table border="1" cellpadding="8"><tbody><tr><td> Reagente </td><td> Importo per 1 reazione </td></tr><tr><td> 10X Buffer Strand Primo </td><td> 2 microlitri </td></tr><tr><td> dNTP Mix </td><td> 4 microlitri </td></tr><tr><td> Inibitore di ribonucleasi </td><td> 1 ml </td></tr><tr><td> Array Script </td><td> 1 ml </td></tr></tbody></table> Tabella 2: Strand cDNA 2 ° Mix sintesi Maestro <table border="1" cellpadding="8"><tbody><tr><td> Reagente </td><td> Importo per 1 reazione </td></tr><tr><td> Acqua priva di nucleasi </td><td> 63 microlitri </td></tr><tr><td> 10X Buffer secondo filone </td><td> 10 microlitri </td></tr><tr><td> dNTP Mix </td><td> 4 microlitri </td></tr><tr><td> DNA polimerasi </td><td> 2 microlitri </td></tr><tr><td> RNasi H </td><td> 1 ml </td></tr></tbody></table> Tabella 3: IVT Master Mix <table border="1" cellpadding="8"><tbody><tr><td> Reagente </td><td> Importo per 1 reazione </td></tr><tr><td> aaUTP soluzione (75 mm) </td><td> 2 microlitri </td></tr><tr><td> ATP, CTP, GTP mix (25 mm) </td><td> 12 microlitri </td></tr><tr><td> T7 soluzione UTP (75 mm) </td><td> 2 microlitri </td></tr><tr><td> T7 tampone di reazione 10X </td><td> 4 microlitri </td></tr><tr><td> T7 Enzyme Mix </td><td> 4 microlitri </td></tr></tbody></table> Tabella 4: Mix ARNA Binding <table border="1" cellpadding="8"><tbody><tr><td> Reagente </td><td> Importo per 1 reazione </td></tr><tr><td> Perline RNA Binding * </td><td> 10 microlitri </td></tr><tr><td> Bead Resuspension * Soluzione </td><td> 4 microlitri </td></tr><tr><td> 100% isopropanolo ** </td><td> 6 microlitri </td></tr><tr><td> ARNA Concentrato Binding Buffer </td><td> 50 microlitri </td></tr></tbody></table> * Mescolare le perline RNA vincolante con la soluzione di risospensione primo tallone. ** Aggiungere l&#39;isopropanolo e mescolare bene prima di aggiungere il legame tampone concentrato ARNA.

<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.

Passaggi critici Un secondo ciclo di amplificazione non è consigliato come pregiudizi nei dati di serie sono stati osservati. Mescolando con cura in ogni fase enzimatica (1 ° e 2 ° sintesi filamento cDNA, IVT) è fondamentale per ottenere buoni rendimenti amplificazione, così come di incubazione di ogni passo enzimatica a temperatura adeguata. Un cycler PCR con regolabile coperchio riscaldato si preferisce - deviazioni anche il più piccolo 2-3 gradi durante IVT in un...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<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>
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vaccinia virus infection &amp; temporal analysis of virus gene expression: part 2

La famiglia<em&gt; Poxviridae</em&gt; È costituito da grandi DNA a doppia elica che contiene virus che si replicano esclusivamente nel citoplasma delle cellule infette. I membri del<em&gt; Orthopox</em&gt; Genere includono vaiolo, l&#39;agente eziologico del vaiolo umano, vaiolo delle scimmie, e vaccinico (VAC), il membro prototipo della famiglia di virus. All&#39;interno del relativamente grande (~ 200 kb) vaccinia genoma, tre classi di geni sono codificati: precoce, intermedio e in ritardo. Anche se tutte e tre le classi sono trascritte dai virale codifica RNA polimerasi, ogni classe svolge una funzione diversa nel ciclo di vita del virus. Poxvirus utilizzano strategie multiple di modulare l&#39;ambiente host cellulare durante l&#39;infezione. Per capire regolamentazione sia di accoglienza e di espressione genica del virus, abbiamo utilizzato tutto il genoma approcci per analizzare abbondanza trascrizione sia da virus e cellule ospiti. In questo studio dimostriamo infezioni corso a tempo di cellule HeLa con il virus vaccinico e RNA di campionamento in diversi momenti dopo l&#39;infezione. Sia host e virale RNA totale è isolato e amplificato per l&#39;ibridazione di microarray per l&#39;analisi dell&#39;espressione genica.

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

Protocollo per vaccinia infezione di cellule HeLa e analisi di accoglienza e l&#39;espressione genica virale. Parte 2 di 3.

Vaccino infezione da virus e analisi temporale di espressione genica del virus: Part 2

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

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8.0K Views.  MIT - Massachusetts Institute of Technology. Protocollo per vaccinia infezione di cellule HeLa e analisi di accoglienza e l'espressione genica virale. Parte 2 di 3.

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

Bacterial Gene Expression Analysis Using Microarrays

Batterica analisi di espressione genica mediante microarray

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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.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

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.

Vaccino infezione da virus e analisi temporale di espressione genica del virus: 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.

Vaccino infezione da virus e analisi temporale di espressione genica del virus: Parte 3

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.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

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.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle 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 ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Ricostituzione in vitro di Active T. castaneum telomerasi

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

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.

Mediata da Agrobacterium Virus-Induced tacere Assay Gene In Cotone

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.

Il MultiBac Protein Complex Piattaforma di produzione al 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.

Analisi globale di RNA Sintesi a livello di singola cellula seguente ipossia

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