第1部分：用Trizol试剂提取总RNA 当工作与RNA是非常重要的工作的环境中，无RNase的。简单的预防措施，如只使用RNA的程序保留移液器和喷涂工作区开始的程序之前，用去污剂试剂（例如，核糖核酸酶客队）是非常有益的。 <ol><li>在1.5 ml离心管要达到一个足够量的RNA池50个斑马鱼的胚胎，并删除尽可能多的水，用移液器。 </li><li>立即加入TRIzol试剂1 250μL到离心管中含有胚胎。 TRIzol试剂含有苯酚，只应根据通风柜使用。 </li><li>裂解及均质颗粒杵（约20杆）的胚胎，直到该组织是充分打乱。 </li><li>当细胞有足够的匀浆，加750μLTRIzol试剂等于1毫升的总量。 </li><li>要允许完整的核蛋白复合物的解离，孵化匀浆样品在室温下5分钟。在这个阶段，样本可闪光灯液氮冷冻和储存于-80 ° C或协议可以继续。 </li><li>要继续加0.2毫升氯仿和15秒，混合岩管。 </li><li>孵育样品在室温下2分钟，然后离心15分钟，在12000 XG 4 ° C。 </li><li>混合物分离成一个较低的红色的酚 - 氯仿相，相间，一种无色的上层水相。上层水相包含的RNA。顶层应包括TRIZOL（约0.6毫升）的初始体积约60％。转移到一个新的离心管，用1毫升吸管小心，不转让任何相间层顶部的水层。 </li><li>沉淀的RNA添加0.5毫升异丙醇。 </li><li>允许样品在室温下坐10分钟，然后离心10分钟样品在12000 XG在4 ° C。 RNA的试管底部形成凝胶颗粒。 </li><li>互不干扰沉淀，去除上清液，用吸管和1毫升75％乙醇洗涤沉淀。混合5分钟的样品轻轻翻转，并在7500 XG离心机在4 ° C。 </li><li>离心后，取出吸管乙醇和样品干倒10分钟时，。 </li><li>加入100μL无RNase水重悬沉淀和孵化的样品在55℃下10分钟。建议经常手指孵化过程中，以帮助RNA的补液的旋涡。或者，您可以使用NanoDrop ND - 1000分光光度计检查样品的质量和数量，或直接继续下一步。 </li></ol>第2部分：RNA清理使用Qiagen公司的RNeasy试剂​​盒 2 Qiagen公司的RNeasy试剂盒是在消除从其他方法中分离出的RNA样品中的污染物和杂质非常有用。对于此过程中，β-巯基乙醇，乙醇，无核酸酶水需要除了在该套件中包含的组件。在此过程中一个可选的DNA酶处理建议。 <ol><li>第一次使用的试剂盒后，缓冲区RPE细胞将需要4卷100％的乙醇加入1体积缓冲区的RPE的准备。 </li><li>在一天的过程中，缓冲器的RLT将需要准备新鲜。计算总额将需要缓冲区的RLT。每个样品350μL的体积需要。要准备的缓冲区的RLT，添加10μLβ-巯基乙醇，每1毫升的缓冲。这项工作应根据通风柜。 </li><li>每个样本缓冲区的RLT 350μL和250μL100％的乙醇添加。拌匀移液器向上和向下几次。 </li><li>转移的样本，应约700μL的RNeasy列是在8000 XG 1分钟，在室温下放置在2毫升收集管和离心机，。 </li><li>离心后，弃流，并添加700μL缓冲液RW1的RNeasy旋转列。 </li><li>离心1分钟，在室温下在8000 XG样本，然后丢弃流过。 </li><li> DNA酶治疗Qiagen公司无RNase的DNA酶试剂盒：DNA酶处理是可选的，但强烈建议提供最高质量的样品。 <ol style="list-style-type:upper-alpha;"><li>当你第一次收到的DNA酶处理工具包，你将需要准备，加入550μL无核酸酶水的DNA酶的DNA酶我原液。轻轻混匀反相和不旋涡。分装成单次使用小瓶含10μL原液。您可以储存在-20 ° C至9个月股市的解决方案。解冻分装在4 ° C的6个星期，如果需要，可以存储，但不重新冷​​冻解冻后的等份。 </li><li>为了准备DNA酶我孵化混合，加入70μL缓冲RDD的10μLDNA酶I S滴答每个样品的解决方案。轻轻颠倒离心管混合简要收集残留的液体形式，管的两侧。直接添加80μLDNA酶我孵化组合的RNeasy旋转列。孵育30分钟，在室温下的DNA酶处理。 </li></ol></li><li>继DNA酶处理，添加350μL缓冲液RW1离心柱和离心1分钟，在室温下在8000 XG。 </li><li>离心后丢弃的流量通过，并添加500μL缓冲的RPE自旋列。孵育样品在室温下5分钟。 </li><li>离心1分钟，在室温下在8000 XG的样品和丢弃的流量通过。 </li><li>离心后加入500μl75％乙醇的离心柱。 </li><li>在8000 XG重复离心，但在室温下2分钟。 </li><li>离心后丢弃的流量，并允许该列干5分钟。 </li><li>立即在室温下离心8000 XG删除任何乙醇的痕迹，左5分钟。 </li><li>离心后是完全转移到一个新的1.5 ml收集管中的自旋列，并添加10μL无核酸酶水。孵育1分钟，然后离心1分钟，在室温下以10,000 XG。的RNA，将在流通过。 </li><li>添加另一种无核酸酶水10μL的离心柱和在室温下孵育1分钟。离心样品在室温下1分钟XG 10,000。 </li><li>离心后取出并丢弃离心柱。 RNA将流通过。 </li><li>检查RNA的数量和质量，按照制造商的说明使用一个NanoDrop ND - 1000分光光度计。 </li><li>此外，运行变性凝胶或使用安捷伦2100生物分析仪检查RNA的完整性。 </li><li>样品应保存于-80℃直至可以进行cDNA合成。 </li></ol>第3部分：使用Invitrogen公司标的第一链系统的cDNA合成 RNA是轻松容易导致退化的核糖核酸酶裂解。因此，许多基因表达的协议，已经发展到使用一个更加稳​​定，已经直接从RNA合成cDNA产物。在这里，我们详细的合成第一链的cDNA用Invitrogen公司标第一链合成系统3。每个cDNA合成反应，最多可容纳5微克的RNA。 <ol><li>在开始的程序组合，并短暂离心​​每个组件。 </li><li>准备RNA /引物混合在无菌的0.5 ml离心管如表1所列。 </li><li>孵育65℃，5分钟的样品。 </li><li> 10分钟，立即转移样品冰浆。 </li><li>当样品冷却准备一个与表2中所述的组件的主结构。卷列出每1反应。总数的反应，相应调整卷。 </li><li>冷却后，加上9日在第5步准备每个RNA /引物混合物的预混液。轻轻混匀后短暂离心收集。每个样品总量应为19μL。 </li><li>在42 ° C孵育2分钟，在热循环样品。 </li><li>标二RT 1μL（50个单位）添加到每个管，混合，并孵育42 ° C为60分钟，在热循环。 </li><li>终止反应，在70 ° C下15分钟，然后寒意样品到4 ° C。步骤8和9可以编程为一个热循环。 </li><li>后的样品已达到4℃，反应转移到冰浴。 </li><li>在冰上，每管加1μl的RNase H的和20分钟在37 ° C。总体积21μL。核糖核酸酶H添加到您的样品会降低RNA为模板，并留下唯一的单链cDNA产物。 </li></ol>第4部分：基因的分离和降水<ol><li> 1.5 ml的锁相硅胶管将用于在隔离的cDNA产品。离心2分钟，在12000 XG准备1.5毫升锁相硅胶管。凝胶都应该在试管底部。 </li><li>锁相凝胶管中加入81.5μL酚（三饱和，缓冲pH值8.0），81.5 24:1的氯仿异戊醇液，样品（21微升）。轻轻颠倒混合数次。 </li><li>在12000 XG离心样品在室温下5分钟。 </li><li> cDNA的，应在上层水相。转移到一个干净的1.5 ml离心管上部阶段。总体积约20μL。 </li><li>添加1X量的3 M NaOAc，pH值5.2，轻轻混匀。例如，如果上层水相的体积等于20μL，您将需要添加2μL3 M NaOAc，pH值5.2。 </li><li>新增7μL5 mg / ml的糖原，轻轻混匀。 </li><li>加入1倍体积的异丙醇，轻轻混匀。一般来说，这是约30μL异丙醇。 </li><li>允许样品在室温temperatur孵育离心12000 XG E为10分钟，然后在室温下20分钟。 </li><li>一个小的cDNA颗粒的试管底部形成。用移液器小心取出上清液。 </li><li>加入500微升70％的乙醇混合反演。 </li><li>离心5分钟，在12000 XG样品。 </li><li>离心后去除上清，用移液器重复的70％乙醇洗涤，离心（步骤10和11）。 </li><li>离心后除去上清液用移液器。如果有液体管双方的剩余，它可以收集简要纺纱管。删除任何多余的液体，使沉淀在室温下干燥5分钟。 </li><li>加入20μL无核酸酶水，补充水分颗粒。 5分钟补液颗粒的样品放置在55 ° C。 </li><li>然后检查以下制造商的说明使用一个NanoDrop ND - 1000分光光度计的cDNA产品的数量和质量。可以储存在-20 ° C的cDNA </li></ol>代表性的成果 RNA的清理后，约15微克的高品质总RNA可以预期从50个斑马鱼的胚胎。为了评估总RNA质量，分光光度仪，凝胶电泳，和Agilent 2100生物分析仪可以使用。一个NanoDrop ND - 1000分光光度计可用于评估在260 nm处的吸光度在280 nm（图1）相比的吸光度。 260 / 280的比例可以揭示污染物的存在，并给予可能的退化的证据。一个260 / 280比例的1.9-2.0认为是可接受的。此外，强烈建议RNA变性凝胶运行评估RNA的完整性。 RNA链将单独对它们的大小的基础上，用较小的股进一步下跌的凝胶迁移。会出现一个非退化的样本作为涂抹两强带。涂片是一个人口大小不同的mRNA链的结果，对应28S和18S rRNA的条带。 28S乐队应该是18S条带（图2）激烈的两倍左右。另外，总RNA的完整性，可以使用Agilent 2100生物分析仪进行评估。两个明显的峰值，代表28S和18S rRNA预期（图3）。 28S峰值应大于18S根据峰面积峰值，​​分别约为2:1。两个明显的峰值，不会退化样品观察。参展降解的RNA样品，不应该通过随后的实验步骤进行。 分光光度分析可用于评估的cDNA产品质量（图4）。 260 / 280应该是1.8左右。输入5μg总RNA的cDNA反应经常在我们的实验室中产生的cDNA产品的1-2微克。 <img src="/files/ftp_upload/1470/1470fig1.jpg" alt="图1" /> 图1。 RNA的清理后的RNA样品NanoDrop的光谱 。，RNA的数量和质量进行评估使用NanoDrop ND - 1000分光光度计。高质量的总RNA中，约15微克， 是常规260 / 280左右1.9-2.0的比例从50斑马鱼的胚胎产生。 <img src="/files/ftp_upload/1470/1470fig2.jpg" alt="图2" /> 图2的RNA变性凝胶要评估的总RNA的分离完整性，RNA变性凝胶可以跑。预计涂抹与相应的28S和18S rRNA的两个明亮的条带。 28S乐队应该是18S乐队激烈的大约两倍。 <img src="/files/ftp_upload/1470/1470fig3.jpg" alt="图3" /> 图3。样本的RNA样品的跟踪，从生物分析仪。评估的总RNA的完整性，可能还可以在Agilent 2100生物分析仪分析。两个尖锐的峰，对应的28S和18S rRNA，应该是可见的的。 28S峰值应大于18S根据峰面积峰值，​​分别约为2:1。 <img src="/files/ftp_upload/1470/1470fig4.jpg" alt="图4" /> 图4。一个NanoDrop频谱的cDNA样本。光度法分析与NanoDrop ND - 1000可用于评估的cDNA产品的数量和质量。 260 / 280的比例应在1.8左右。输入5微克总RNA的cDNA反应，经常产生1-2微克的cDNA在我们的实验室。 表1。元件RNA /引物混合物添加在合成cDNA的第2步。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 组件 </td><td> 卷 </td></tr><tr><td>总RNA（5微克） </td><td> 10μL </td></tr><tr><td> 10毫米dNTP混合液</td><td> 1μL </td></tr><tr><td>以oligo（dT）12-18（0.5μg/μL的） </td><td> 1μL </td></tr><tr><td> DEPC处理过的水</td><td> ñμL </td></tr><tr><td>总成交量</td><td> 10μL </td></tr></tbody></table> 表2。cDNA合成的第5步反应混合物。卷列出的每个反应。每个组件数量增加总数的反应。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 组件 </td><td> 卷 </td></tr><tr><td> 10X RT缓冲</td><td> 2μL </td></tr><tr><td> 25 mM的氯化镁</td><td> 4μL </td></tr><tr><td> 0.1 mM的数码地面电视</td><td> 2μL </td></tr><tr><td> RNAseOUT </td><td> 1μL </td></tr></tbody></table>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> TRIzol Reagent Manual. , (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RNEasy Mini Handbook. , (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SuperScript First-Strand Synthesis System for RT-PCR. , (2003).</li></ol>

The authors have nothing to disclose.

RNA分子的脆弱性是最重要的考虑因素，应成为本议定书记住。应始终使用无菌设备，无RNase在实验室无RNase区。储备的只适用于RNA的程序实验室的一部分，它是有帮助的。核糖核酸酶去除产品，如核糖核酸酶，远离此区域应经常喷洒。 RNA样本应始终带手套处理，并放在冰上，防止退化。 该协议利用市售的试剂和试剂盒。另外，还有许多额外的试剂和RNA分离试剂盒，市场上可?...

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		<div>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Glycogen (5 mg/ml)</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>9510</td>
<td>&nbsp;</td>
<tr>
<td>NanoDrop ND-100</td>
<td>&nbsp;</td>
<td>Thermo Scientific </td>
<td>ND-1000</td>
<td>&nbsp;</td>
<tr>
<td>Pellet Pestles with Microfuge Tube</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>KT749520-0090</td>
<td>&nbsp;</td>
<tr>
<td>Phase Lock Gel Light, 1.5 ml</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>2302800</td>
<td>&nbsp;</td>
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<td>Phenol, Tris-saturated</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>3117944001</td>
<td>&nbsp;</td>
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<td>RNAse Away</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>17810-491</td>
<td>&nbsp;</td>
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<td>RNAse-Free DNase Set</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>79254</td>
<td>&nbsp;</td>
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<td>RNEasy Mini Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>74104</td>
<td>&nbsp;</td>
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<td>SuperScript&reg; First-Strand Synthesis System for RT-PCR</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11904-018</td>
<td>&nbsp;</td>
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<td>TRIzol</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15596-026</td>
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rna isolation from embryonic zebrafish and cdna synthesis for gene expression analysis

许多重要和复杂的实验室的程序，要求输入的高品质，完整的RNA。降级样品或杂质的存在可能导致灾难性的结果在下游的实验应用。因此，最重要的，使用坚实的技术与众多的保障措施和质量控制检查，以确保卓越的样本。在此，我们详细的协议隔离使用市售的化学变性和随后的清理，以去除DNA和使用商业RNA提取试剂盒杂质的痕迹，从整个斑马鱼胚胎的总RNA。作为RNA相对不稳定和轻松容易由核糖核酸酶裂解，大多数协议检测基因表达的cDNA的产品，是直接从RNA为模板合成。我们详细的程序转换成更稳定的cDNA使用市售的套件产品的RNA。纵观这些程序有多项质量控制检查，以确保样品不退化或受到污染的的。这些协议的最终产品是基因，是适合的芯片分析，RT - PCR或长期储存。

Watch this Scientific Journal Video about RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis at JoVE.com

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

孤立的高品质，完整的RNA是一个必不可少的步骤在许多实验室的协议。在这里，我们从整个斑马鱼的胚胎，随后应用在各种实验程序，包括基因表达芯片分析cDNA合成RNA的提取证明。

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

rna-isolation-from-embryonic-zebrafish-cdna-synthesis-for-gene

Research

JoVE Journal

Biology

37.7K 次观看.  Purdue University. 孤立的高品质，完整的RNA是一个必不可少的步骤在许多实验室的协议。在这里，我们从整个斑马鱼的胚胎，随后应用在各种实验程序，包括基因表达芯片分析cDNA合成RNA的提取证明。

视频: RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

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

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 ...

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 ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

从小鼠胚胎肾肾区细胞的分离和培养

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

高分辨率基因表达谱RNA的合成，加工和衰变的新转录RNA在细胞培养的代谢标记

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

从解离分析准备胚胎和幼体斑马鱼骨肌纤维

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

标记的细胞核从亲和力为基础的隔离<em&gt;果蝇</em&gt;组织的基因表达分析

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

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 ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

有针对性的RNA测序分析表征基因表达和基因组改变

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...