Name: rna isolation from embryonic zebrafish and cdna synthesis for gene expression analysis
Uploaded: 2009-08-07T16:40:00
Description: Watch this Scientific Journal Video about RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis at JoVE.com

<p class="jove_title">第1部分：用Trizol试剂提取总RNA </p><p class="jove_content">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。 </p><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<sup>1</sup> 250μL到离心管中含有?...

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	<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> <em style='font-style: italic'>TRIzol Reagent Manual</em>. , (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> <em style='font-style: italic'>RNEasy Mini Handbook</em>. , (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> <em style='font-style: italic'>SuperScript First-Strand Synthesis System for RT-PCR</em>. , (2003).</li></ol>

<p class="jove_content"> 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。 </p><p class="jove_content">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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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
<tr>
<td>Phenol, Tris-saturated</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>3117944001</td>
<td>&nbsp;</td>
<tr>
<td>RNAse Away</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>17810-491</td>
<td>&nbsp;</td>
<tr>
<td>RNAse-Free DNase Set</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>79254</td>
<td>&nbsp;</td>
<tr>
<td>RNEasy Mini Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>74104</td>
<td>&nbsp;</td>
<tr>
<td>SuperScript&reg; First-Strand Synthesis System for RT-PCR</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11904-018</td>
<td>&nbsp;</td>
<tr>
<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

<p class="jove_content">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。</p>

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

<p class="jove_content">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。</p>

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 ...

Research

JoVE Journal

Biology

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

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

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

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

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

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

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

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 <em>Drosophila</em> Tissues for Gene Expression Analysis

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

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

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

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 ...