怀特黑德研究所的研究员基金

第1部分：从RNA合成cDNA <ol><li>使用Ambion公司氨基烯丙基MessageAmp II套件之前，建议量的100％的乙醇添加到洗缓冲区。 </li><li>在PCR反应管中，加之间100ng的总RNA，以oligo（dT）引物的T7加入1μl5μg。把音量无核酸酶水至12μl。 </li><li>孵育样品在70℃，在热循环10分钟。 </li><li>删除从70℃，短暂离心的RNA样品。置于冰上。 </li><li>准备的第一链合成预混，并保持在室温（10％的移液错误的盘盈）（见表 1）。 </li><li>轻轻吸预混或轻弹混合，然后短暂离心。 </li><li>将主结构8μl转移到每个RNA样品。混合移液器向上和向下的3-4倍。 </li><li>在42 ° C孵育2小时在热循环。 </li><li>离心机样本简要置于冰上。立即进行下一步的双链DNA合成。 </li><li>准备在冰第二链的合成大师的组合（10％的移液错误的盘盈）（表 2） </li><li>轻轻吸预混或轻弹混合，然后短暂离心。 </li><li>每个样品转移80μl，轻轻混匀移液器向上和向下的3-4倍。 </li><li>在16℃孵育2小时在热循环。 </li><li>经过2个小时的潜伏期，进行cDNA的清理步骤，或立即冻结在-20 ° C。 </li></ol>第2部分：双链cDNA清理<ol><li>从冰箱中取出的cDNA纯，并允许它在使用前30分钟平衡至室温。摇动瓶子在使用前要充分重悬磁cDNA的约束力珠。 </li><li>分装成1.5ml离心管，并在50-60 ° C孵育至少10分钟，在过去的2小时潜伏期无核酸酶水。 </li><li>每个样品添加纯180μl的cDNA，并彻底混合向上和向下吹打。样品转移到一个96孔的圆底板。 </li><li>继续混合样品至少2分钟，轻轻晃动轨道摇床板。 </li><li>移动板磁性的立场，以捕获磁珠。留在板约6分钟的立场，或直到混合物变得透明和结合珠颗粒。 </li><li>小心吸上清液真空抽吸，而不会干扰磁珠。另外，用移液器小心取出上清，弃上清。 </li><li>删除从磁立场板。 </li><li>加入150μlcDNA的洗涤缓冲液，每孔摇动1分钟，速度适中的轨道摇床板。珠不会分散在这一步，由于洗涤缓冲液的表面张力低。 </li><li>移动板磁性的立场，以捕获磁珠。 </li><li>小心吸上清液真空抽吸，而不会干扰磁珠。另外，用移液器小心取出上清，弃上清。 </li><li>删除从磁立场板。 </li><li>重复与150μlcDNA的洗涤缓冲液洗2 次的时间。 </li><li>之后的第2 次洗，干燥摇晃以最快的速度为2分钟轨道摇床板的珠子。不要overdry！ </li><li>每个样品加入18μl预热无核酸酶水洗脱珠的cDNA。 </li><li>大力动摇盘3分钟轨道摇床，然后检查，以确保磁珠完全散去。如果没有，继续摇晃。 </li><li>一旦磁珠完全散去，移动板磁性的立场，以捕获磁珠。 </li><li>小心地转移到一个新的PCR板（或PCR管）洗脱的cDNA（〜16μl）。 </li><li>直接进入下一步，或冻结在-20 ° C的cDNA </li></ol>第3部分： 体外转录（IVT） <ol><li>准备在室温下的IVT 预混（见表3 ） </li><li>轻轻吸液管硕士的组合或轻弹混合，并短暂离心​​。 </li><li>每个样品中加入24μl，轻轻混匀移液器向上和向下的3-4倍。 </li><li>在37 ° C孵育14小时的热循环，然后保持在4 ° C，直到为下一步做好准备。 </li></ol>第4部分：ARNA干净后IVT <ol><li>旋涡RNA结合珠简要地获取，使用前均匀混合。 </li><li> （见表4）这是可以做到提前准备ARNA绑定混合，在室温下-可以在室温下储存长达一个星期的准备约束力混合。 </li><li>由涡旋混合。 </li><li>分装成1.5ml离心管，并在50-60℃孵育至少10分钟的ARNA洗脱缓冲液。 </li><li>添加ARNA约束力混合70μl每个样品中，拌匀移液器向上和向下的3-4倍。 </li><li>样品PCR板转移到一个96孔的圆底板。 </li><li> 100％异丙醇每个样品中加入50μL拌匀移液器向上和向下的3-4倍。 </li><li>轻轻摇动至少2分钟，轨道摇床板，彻底混合样品。 </li><li>移动板磁性的立场，以捕获磁珠。离开盘上的立场，直到混合物变得透明和约束力珠颗粒。 </li><li>小心吸上清液真空抽吸，而不会干扰磁珠。另外，用移液器小心取出上清，弃上清。 </li><li>删除从磁立场板。 </li><li>加入100μLARNA每口井的清洗液，摇动1分钟，速度适中的轨道摇床板。珠可能无法完全分散在这一步。 </li><li>移动板磁性的立场，以捕获磁珠。 </li><li>小心吸上清液真空抽吸，而不会干扰磁珠。另外，用移液器小心取出上清，弃上清。 </li><li>删除从磁立场板。 </li><li>重复100μLARNA清洗液洗了第二 。 </li><li>之后的第2 次洗，干燥摇晃以最快的速度为1分钟的轨道摇床板的珠子。不要overdry的样品！ </li><li>每个样品加入40μl预热ARNA洗脱液洗脱珠ARNA。 </li><li>大力动摇盘3分钟轨道摇床，然后检查，以确保磁珠完全散去。如果没有，继续摇晃。 </li><li>一旦磁珠完全散去，移动板磁性的立场，以捕获磁珠。上清包含清理氨基烯丙基纳入ARNA。 </li><li>洗脱ARNA小心地转移到一个新的PCR板（或PCR管）。 </li><li> （可选步骤）检查上NanoDrop分光光度计测量1.5μlRNA浓度的样品。 </li><li>立即继续到下一个步骤，或存放于-80℃的ARNA ° C。 </li></ol>表1：cDNA的第一链合成预混<table border="1" cellpadding="8"><tbody><tr><td> 试剂 </td><td> 金额为1反应 </td></tr><tr><td> 10X第一链缓冲</td><td> 2μL </td></tr><tr><td> dNTP混合液</td><td> 4μL </td></tr><tr><td>核糖核酸酶抑制因子</td><td> 1μL </td></tr><tr><td>阵列脚本</td><td> 1μL </td></tr></tbody></table>表2：cDNA的第二链合成预混<table border="1" cellpadding="8"><tbody><tr><td> 试剂 </td><td> 金额为1反应 </td></tr><tr><td>核酸免费水</td><td> 63μL </td></tr><tr><td> 10X第二链缓冲</td><td> 10μL </td></tr><tr><td> dNTP混合液</td><td> 4μL </td></tr><tr><td> DNA聚合酶</td><td> 2μL </td></tr><tr><td>核糖核酸酶H </td><td> 1μL </td></tr></tbody></table>表3：IVT中的master mix <table border="1" cellpadding="8"><tbody><tr><td> 试剂 </td><td> 金额为1反应 </td></tr><tr><td> aaUTP解决方案（75毫米） </td><td> 2μL </td></tr><tr><td> ATP，CTP，GTP的组合（25毫米） </td><td> 12μL </td></tr><tr><td> T7 UTP解决方案（75毫米） </td><td> 2μL </td></tr><tr><td> T7 10X反应缓冲液</td><td> 4μL </td></tr><tr><td> T7酶混合物</td><td> 4μL </td></tr></tbody></table>表4：ARNA绑定混合<table border="1" cellpadding="8"><tbody><tr><td> 试剂 </td><td> 金额为1反应 </td></tr><tr><td>核糖核酸*装订珠</td><td> 10μL </td></tr><tr><td>珠再悬浮解决方案* </td><td> 4μL </td></tr><tr><td> 100％的异丙醇** </td><td> 6μL </td></tr><tr><td> ARNA绑定缓冲液</td><td> 50μL </td></tr></tbody></table> *混合RNA结合珠与珠再悬浮溶液。 **添加异丙醇和拌匀之前加入ARNA结合缓冲液。

<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.

关键步骤一个放大的第二轮，是不是表示已经观察到数组中的数据的偏差。仔细搅拌在每个酶一步（ 第一和第二链cDNA合成，IVT），取得了良好的扩增产量的关键，是在适当的温度孵化每个酶一步。一个可调节的热盖的PCR基因扩增仪是首选 - 甚至偏差，在IVT中为2-3度，空气杂交烘箱或水浴杂交小，可以显着影响产量的扩增产物。 应用程序/意?...

<!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>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

家庭<em&gt; Poxviridae</em&gt;由大型双链DNA含有病毒在感染细胞的细胞质中完全复制。成员<em&gt;痘</em&gt;属包括天花，人类的小痘病原体，猴痘，牛痘（VAC）的病毒家族的原型成员。在比较大（约200 KB）痘苗病毒基因组中，3类基因编码的早期，中期，和后期。维拉利编码RNA聚合酶转录虽然所有这三个类，每一类有一个病毒的生命周期中的不同功能。痘病毒利用调制主机在感染的细胞环境的多种策略。为了了解主机和病毒基因表达的调控，我们利用全基因组的方法，从病毒和宿主细胞的转录丰度分析。在这里，我们展示日久感染的HeLa细胞在几个时间点后感染牛痘病毒和采样RNA。主机和病毒总RNA分离和扩增为杂交微阵列基因表达分析。

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

协议牛痘感染的HeLa细胞和宿主和病毒基因的表达分析。

牛痘病毒感染及病毒基因表达的时空分析：第2部分

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 次观看.  MIT - Massachusetts Institute of Technology. 协议牛痘感染的HeLa细胞和宿主和病毒基因的表达分析。

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

Bacterial Gene Expression Analysis Using Microarrays

使用微阵列的细菌基因表达分析

科研

生物学

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.

主电穿孔的小鼠骨髓造血细胞培养制备

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.

牛痘病毒感染及病毒基因表达的时空分析：第1部分

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.

牛痘病毒感染及病毒基因表达的时空分析：第3部分

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.

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

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.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

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.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

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.

农杆菌介导的病毒诱导的基因沉默含量在棉花

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.

在EMBL的MultiBac蛋白质复杂的生产平台

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.

全球合成的RNA在单细胞水平以下的低氧分析

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