逆转录反应关闭加热块到70-80 ° C和42 ° C。水浴也可以用在这一步。如果使用加热块，放一些水，使热量均匀。 <ol><li>底漆反应<ol><li>取3微克的RNA </li><li>调整音量与核糖核酸酶自由水至13μL </li><li>加入2.5μL的六聚体随机引物（2mg/mL） </li></ol></li><li>搅拌均匀，在70-80℃孵育8分钟。然后，放置在冰浴5分钟。 </li><li>准备RT鸡尾酒（14.5μL/ RXN）： <table border="0"><tbody><tr><td> 组件 </td><td> 卷 </td></tr><tr><td> 5X第一链缓冲</td><td> 6μL </td></tr><tr><td> 0.1中号数码地面电视 </td><td> 3μL </td></tr><tr><td> 25X aminoallyl dNTP混合液</td><td> 1.2μL </td></tr><tr><td>标II RT（200U/μl） </td><td> 2.3μL </td></tr><tr><td>水 </td><td> 2.0μL </td></tr></tbody></table> * n个反应，准备为n等分0.5鸡尾酒法师组合，以保证你不会用完。 </li><li>加入14.5μL冷却反应。 </li><li>混合（不要旋涡），并在42 ° C孵育3-4小时</li><li>如有必要，样品可以储存在-20 ° C。 </li></ol>水解的RNA <ol><li>每个样品中，添加3μL的1 N NaOH和0.6μL0.5 M EDTA。 （最后conc.s = 100MM 10MM EDTA的氢氧化钠，） </li><li>混合并孵育65 ° 10分钟。 </li><li>加入33.6μL1M HEPES中性，pH值= 7.0（终浓度.= 500MM HEPES） </li><li>样品可以储存在-20 ° C，如有必要。 </li></ol>反应净化：清除非法人AA -的dUTP和自由胺 注 ：此净化协议是从Qiagen公司的QIAquick PCRpurification套件协议的修改。 Qiagen公司提供的缓冲区，因为Qiagen公司的缓冲区包含自由竞争与CY染料偶联反应的胺取代磷酸盐的洗涤和洗脱缓冲液。也可用于从小型预习套件列。 <ol><li> 300μL（或5X反应体积= 336μL）缓冲液PB（QIAGEN提供）混合cDNA反应，并转移到QIAquick列。 </li><li>将在2 ml收集管中柱（QIAGEN提供）离心1分钟〜13000 RPM。清空收集管。 </li><li>要洗净，加750μL磷酸盐缓冲液洗（不Qiagen公司）列和离心1分钟〜13000 RPM。 </li><li>清空收集管，重复750μL洗涤和离心步骤。 </li><li>清空收集管，并以最大的速度增加1分钟离心列。 </li><li>列转移到一个新的1.5 ml离心管，小心地添加30μL磷酸盐洗脱缓冲液。 （见溶液的制备部分） </li><li>孵育1分钟室温。 </li><li> 1分钟〜13000 RPM离心洗脱。 </li><li>第二次（与其他磷酸盐洗脱缓冲液30μL）重复步骤6-8到同一管流出。最终洗脱体积应〜60μL。 </li><li>在这个阶段，可以量化的cDNA量： 吴的cDNA =（稀释倍数）（OD260）（37）（总量列洗脱） </li><li>干样的速度在45 VAC ° C。 </li><li>如有必要，样品可以储存在-20 ° C。 </li></ol>耦合AA基因CY染料酯<ol><li>悬浮aminoallyl标记的cDNA 10μL50毫米钠碳酸氢盐，pH值= 9.0，如果必要的。 （此缓冲区应该是新鲜，每两个星期） </li><li>在黑暗的地方工作，加上适当的Cy染料管探头。吸取最多几次悬浮染料。 </li><li>反应在室温避光孵育1小时。 （孵化可以去两个小时） </li></ol>反应净化：去除耦合染料<ol><li>添加35μL100毫米NaOAc pH值5.2的反应。调匀。 </li><li>加入250μL（或5X反应体积= 225μL）缓冲区PB（QIAGEN提供）上下吹打每个样品和混合。 </li><li>将在2毫升收集管QIAquick离心柱（QIAGEN提供），适用于样品列，离心1分钟〜13000。清空收集管。 </li><li>要洗净，加0.75 mL缓冲液PE（QIAGEN提供）1分钟〜13000列和离心机。 </li><li>重复步骤4。 </li><li>空集合管和离心机以最大的速度增加1分钟的列。 </li><li>地点列在一个干净的1.5 ml离心管，小心地加入30μl的缓冲液EB（QIAGEN提供）列膜的中心。 </li><li>孵育1分钟室温。 </li><li> 1分钟〜13000 RPM离心洗脱。 </li><li>重复第二次到同一管流出步骤7-9。最后的洗脱体积应〜60μL。 </li></ol>标记反应分析在这个阶段可以量化的cDNA量和标签： <ul><li>测量外径260，550和650（空白是水。）使用nanodrop的芯片方案。 </li><li>计算pmol染料掺入pmol的DNA与国统会/染料的比例给予下面公式： pmol核苷酸= [OD260 *量（μl）* 37毫微克/μL* 1000皮克/ NG] / 324.5 PG / pmol </li></ul> 注 ：1 OD260 = 37纳克/μLcDNA的; 324.5 pg / pmol的一个dNTP的平均分子量） pmol Cy3标记= OD550 *量（μl）/ 0.15 pmol Cy5的量（μl）/ 0.25 = OD650 核苷酸/染料比率= pmol的cDNA / pmol赛扬染料如果您正在使用nanodrop的，它已经给每个染料pmol /μL。你只需要这个数字乘以数量得到总pmo​​l染料掺入。 注 ：&gt; 150-200 pmol染料掺入每个样品的比例小于20个核苷酸/染料分子杂交的最佳选择。 <ul><li>在黑暗的样品可以储存在-20 ° C。 <ul><li>结合探针杂交在一起，干倒在Speed​​Vac在42 ° C。 </li><li>样品可以储存在-20 ° C，直到准备来HYB。 </li></ul></li></ul>前HYB幻灯片<ol><li>放置滑面，跌幅超过RT 100毫升水3-5分钟，在一个干净的红色幻灯片持有人</li><li>对齐干，放置在100℃加热块面对几秒钟的幻灯片 - 手表凝结消失</li><li>紫外光交联在250 mJoules幻灯片（紫外线交联剂输入2500） </li><li>预热42℃的热水中浸渍水合物的幻灯片。旋转700 RPM在室温5分钟。 </li><li>孵育于50ml Coplin JAR幻灯片包含至少45分钟的预热预HYB（5XSSC，1％BSA，0.1％SDS）@ 42 ° C，并且潜伏期可以去如果必要的时间。 50毫升HYB前缓冲区：12.5毫升20X SSC，10毫升5％BSA，0.5毫升10％SDS，27 mL水 </li><li> DIP幻灯片和预热水（42℃）搅拌几分钟删除前HYB缓冲区。 </li><li>异丙醇冲洗和旋转700 RPM，室温，干燥5分钟。 </li></ol>准备样品杂交<ol><li>悬浮在水中的探头（卷下面给出） <table border="0"><tbody><tr><td>对于总卷。 =30μl </td><td>对于总卷。 =35μl </td><td>总卷.=40μl </td><td></td></tr><tr><td> 19.8μL水 </td><td> 23.55μL水 </td><td> 27.2μL水 </td><td> </td></tr><tr><td> 1.5μL </td><td> 1.5μL </td><td> 1.5μL </td><td> 10毫克/毫升的鲑鱼精子DNA（Invitrogen公司） </td></tr><tr><td> 1.2μL </td><td> 1.2μL </td><td> 1.2μL </td><td> 12.5毫克/毫升的tRNA </td></tr><tr><td> 4.5μL </td><td> 5.25μL </td><td> 6μL </td><td> 20XSSC（3X最后） </td></tr><tr><td> 3μL </td><td> 3.5μL </td><td> 4μL </td><td> 1％SDS </td></tr></tbody></table> 注 ：此外试剂的顺序是很重要的。不要准备预混。 </li><li>煮沸5分钟，然后旋转14000转5分钟。室温2分钟冷却。 </li></ol>杂交<ol><li>加入10-12μl的3倍SSC的每个边缘和中间的杂交室井。 </li><li>添加清洁升降机滑幻灯片的适当区域，并在杂交液轻轻加载（30 -40μL）。应该有额外的样品任盖玻片边缘。 </li><li>放置在会议厅内的幻灯片。拧紧螺钉和​​地方在65℃水浴过夜。不要直接放在水浴底部的腔。至上腔顶部放置一个块保存一切就绪。继续水浴盖子保护光敏感的样品。 </li></ol> HYB洗<ol><li>准备以下的缓冲区： <ul><li> 1X SSC，0.05％SDS </li><li> 0.06 X SSC </li></ul></li><li>孵化后启封杂交室。从试验箱中取出的幻灯片，小心不要打扰盖玻片。 </li><li>要删除盖玻片，淹菜中含有温暖1X SSC，0.05％SDS的缓冲液洗盖玻片面临的菜底部的幻灯片。盖玻片会脱落移动幻灯片一边到另一边。 </li><li>盖玻片后取出，放置在一个温暖1XSSCm 0.05％SDS的机架新鲜浴的幻灯片。鼓动几分钟</li><li>将幻灯片加上机架0.06 X SSC洗澡。 </li><li>幻灯片传输到另一个BA日的0.06 X SSC包含一个新的机架，在纸巾上的印迹幻灯片之前删除尽可能多的SDS含缓冲区尽可能放置在新鲜浴。 </li><li>搅拌几分钟的幻灯片。 </li><li>立即在室温下自旋为5分钟700转，然后幻灯片准备扫描。商店在黑暗中，直到扫描。 </li></ol>

<ol>
	<li>Hasseman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TIGR+Aminoallyl+Labeling+of+RNA+for.">TIGR Aminoallyl Labeling of RNA for.</a> Microarrays &amp; TIGR Microarray Labeled Probe Hybridization. , .</li><li>Gilbert, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TIGR+Microbial+RNA+Aminoallyl+Labeling+for+Microarrays+&amp;+Hybridization+of+probed+labels.">TIGR Microbial RNA Aminoallyl Labeling for Microarrays &amp; Hybridization of probed labels.</a> , .</li><li>Hedge, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+concise+guide+to+cDNA+Microarray+analysis-II.">A concise guide to cDNA Microarray analysis-II.</a> , .</li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>AA-dUTP</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A0410</td>
<td>5-(3-aminoallyl)-2&#x2019;deoxyuridine-5&#x2019;-triphosphate</td>
</tr>
<tr>
<td>dNTP</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>27-2035-01</td>
<td>100 mM dNTP Set PCR grade</td>
</tr>
<tr>
<td>Random Hexamer primers</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>27-2166-01</td>
<td>3mg/mL</td>
</tr>
<tr>
<td>SuperScript III RT</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>80800444</td>
<td>200U/&#x03BC;L</td>
</tr>
<tr>
<td>CyDye&#x2122;</td>
<td>&nbsp;</td>
<td>Amersham</td>
<td>RPN5661</td>
<td>Post-Labelling Reactive Dye Pack</td>
</tr>
<tr>
<td>QIAquick</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28104</td>
<td>PCR Purification kit</td>
</tr>
<tr>
<td>1M K2HPO4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1M KH2PO4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1M KPO4</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To make a 1M Phosphate buffer (KPO4, pH 8.5-8.7) combine:
9.5 mL 1M K2HPO4 and 0.5 ml 1M KH2PO4
</td>
</tr>
<tr>
<td>Phosphate wash buffer</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For 100 mL Phosphate wash buffer (5 mM KPO4, pH 8.0, 80% EtOH) mix: 0.5 ml 1 M KPO4 pH 8.5 + 15.25 mL MilliQ water + 84.25 mL 95% ethanol. Wash buffer will be slightly cloudy.
** IMPORTANT: Phosphate wash buffer should be prepared daily.</td>
</tr>
<tr>
<td>Phosphate elution buffer</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Diluting 1 M KPO4, pH 8.5 to 4 mM with sterile water</td>
</tr>
<tr>
<td>Sodium Carbonate Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>(Na2CO3): 0.5M, pH 9.0. Dissolve 4.2 g NaHCO3 in 80 mL of sterile water and adjust pH to 9.0 with 10 N NaOH; bring volume up to 100 mL with sterile water. To make a 50 mM solution for the dye coupling reaction dilute 1:10 with water.
Note: Carbonate buffer changes composition over time; make it fresh every couple of weeks to a month.</td>
</tr>		</tbody>
		</table>
		</div>

bacterial gene expression analysis using microarrays

Watch this Scientific Journal Video about Bacterial Gene Expression Analysis Using Microarrays at JoVE.com

Bacterial Gene Expression Analysis Using Microarrays

使用微阵列的细菌基因表达分析

bacterial-gene-expression-analysis-using-microarrays

Research

JoVE Journal

Biology

8.7K 次观看.  University of California Santa Cruz - UCSC. 

视频: Bacterial Gene Expression Analysis Using Microarrays

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

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 ...

科研

生物学

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

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

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

牛痘病毒感染及病毒基因表达的时空分析：第3部分

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

全球基因表达分析，使用斑马鱼的寡核苷酸芯片平台

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

Spinal Cord Electrophysiology

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

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

脊髓电

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

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

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 ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

白蜡窄吉丁的基因表达分析（ Agrilus planipennis）使用实时定量PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

（充电）酰化tRNA的使用微阵列技术水平的全基因组分析

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

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

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

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

石蜡包埋及冷冻的部分果蝇成人肌肉

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