Name: sample preparation and analysis of rnaseq-based gene expression data from zebrafish
Uploaded: 2017-10-27T12:30:00
Description: Watch this Scientific Journal Video about Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish at JoVE.com

这项工作得到了 R01DK102001 (N.A.Z.)、P30DK072488 (N.A.Z.) 和 T32DK098107 (T.L.H. 和 J.E.N.) 的支持。

下面概述的所有动物协议均符合并经马里兰大学动物保育和使用委员会 (IACUC) 批准. 
 1. 胚准备 <ol> <li> 通过自然交配生成胚胎 <ol> <li> 培养胚胎到3月龄, 生殖成熟度 5 , 8.</li>
		<li> 在胚胎采集前, 将成年雄性和雌性鱼从所需的菌株分离到分离的交配槽中, 并在每个容器中添加2雄性和3雌性. 
		注: 使用转基因的 <e...

<ol>
	<li>Nusslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish. , (2002).</li><li>Detrich, H. W., Zon, L., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish: Disease Models and Chemical Screens. , (2017).</li><li>Detrich, H. W., Zon, L. I., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish: Genetics, Genomics, and Transcriptomics. , (2016).</li><li>Detrich, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish: Genetics, Genomics and Informatics. , (2011).</li><li>Avdesh, A., 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=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> J Vis Exp. (69), e4196 (2012).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), (2009).</li><li>Hwang, W. Y., 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=Efficient+genome+editing+in+zebrafish+using+a+CRISPR-Cas+system.">Efficient genome editing in zebrafish using a CRISPR-Cas system.</a> Nat Biotechnol. 31 (3), 227-229 (2013).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Rossi, A., 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=Genetic+compensation+induced+by+deleterious+mutations+but+not+gene+knockdowns.">Genetic compensation induced by deleterious mutations but not gene knockdowns.</a> Nature. 524 (7564), 230-233 (2015).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>Samsa, L. A., Fleming, N., Magness, S., Qian, L., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Single+Cells+from+Zebrafish+Embryos.">Isolation and Characterization of Single Cells from Zebrafish Embryos.</a> J Vis Exp. (109), (2016).</li><li>. IDT Primerquest Tool Available from: <a target="_blank" href="https://www.idtdna.com/Primerquest/Home/Index">https://www.idtdna.com/Primerquest/Home/Index</a> (2017)</li><li>Heid, C. A., Stevens, J., Livak, K. J., Williams, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+quantitative+PCR.">Real time quantitative PCR.</a> Genome Res. 6 (10), 986-994 (1996).</li><li>Leitch, C. C., Lodh, S., Prieto-Echague, V., Badano, J. L., Zaghloul, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+body+proteins+regulate+Notch+signaling+through+endosomal+trafficking.">Basal body proteins regulate Notch signaling through endosomal trafficking.</a> J Cell Sci. 127 (Pt 11), 2407-2419 (2014).</li><li>Lodh, S., Hostelley, T. L., Leitch, C. C., O'Hare, E. A., Zaghloul, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+on+beta-cell+mass+by+disruption+of+Bardet-Biedl+syndrome+or+Alstrom+syndrome+genes.">Differential effects on beta-cell mass by disruption of Bardet-Biedl syndrome or Alstrom syndrome genes.</a> Hum Mol Genet. 25 (1), 57-68 (2016).</li><li>Hostelley, T. L., Lodh, S., Zaghloul, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+organism+transcriptome+analysis+of+zebrafish+models+of+Bardet-Biedl+Syndrome+and+Alstrom+Syndrome+provides+mechanistic+insight+into+shared+and+divergent+phenotypes.">Whole organism transcriptome analysis of zebrafish models of Bardet-Biedl Syndrome and Alstrom Syndrome provides mechanistic insight into shared and divergent phenotypes.</a> BMC Genomics. 17, 318 (2016).</li></ol>

本协议中描述的方法提供了一个相对快速和 cost-effective 的策略, 用于对整个动物或特定排序的细胞群进行转录分析。斑马鱼为这种类型的研究提供了一个有利的模式, 因为在产生大量的起始物质, 易于实施遗传或环境实验条件, 以及大量的可用性光谱的转基因记者线允许隔离细胞类型的特定和组织特定的群体。
虽然这里没有详细说明, 但这种方法可以很容易地容纳从幼鱼或成年...

全球基因表达的比较分析是鉴别新基因的重要工具。这种分析通常依赖于对实验和对照样本的成绩单丰度进行定量评估。靶向方法, 如 qRT PCR 是相对快速和准确的调查单基因表达变化。RNA 测序 (RNASeq) 提供了一种宽泛的、无假设的方法来识别样本间基因表达的显著变化, 使之成为跨实验系统进行此类调查的标准。
斑马鱼已成为一个突出的模式, 在许多疾病地区。最初为他们的用途开发生物学研究, 由于他们的高繁殖力和相对地低维护费用, 试验性使用斑马鱼已经演变包括各种各样的表型从胚胎到成人阶段并且分子分析的宽数组1,2,3。事实上, 这些优势使分子机械研究迅速和 cost-effective, 因为容易获得大量的材料结合在一起, 在生活的所有阶段的遗传和环境操作的容易。此外, 斑马鱼的胚胎和幼虫的透明性质使它非常适合于生成细胞和组织特异的转基因报告线, 允许在体内离散细胞群的可视化4。利用此类线, 根据报告基因表达, 对特定孤立细胞类型进行全球基因表达分析。
在这里, 我们提出了一个全面的协议, 全球基因表达分析使用 RNASeq 后, 养殖斑马鱼胚胎。基因实验操作, 包括基于吗 (MO) 的瞬态基因击倒或 CRISPR 介导的基因组编辑, 已在别处出现5,6,7。因此, 我们专注于一个详细的协议, 分离 RNA 从整个胚胎或排序的转基因记者表达细胞后, 简单的计算分析 RNASeq 结果使用路径工具和基因本体 (GO) 术语。最后, 我们还包括了一种通过定量逆转录酶 pcr (qRT pcr) 验证基因表达变化的策略。这些协议适用于斑马鱼的胚胎在广泛的实验条件, 包括比较遗传突变体或环境条件。

<table>
	<tbody>
		<tr>
			<td>Commercial Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TriZol</td>
			<td>Thermo Scientific</td>
			<td>15596026</td>
			<td>lysis reagent</td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td>dissociation buffer 1</td>
		</tr>
		<tr>
			<td>FACSMax</td>
			<td>Genlantis</td>
			<td>T200100</td>
			<td>dissociation buffer 2</td>
		</tr>
		<tr>
			<td>DEPC-treated water</td>
			<td>Sigma</td>
			<td>95284</td>
			<td></td>
		</tr>
		<tr>
			<td>FirstStrand cDNA conversion</td>
			<td>Thermo Scientific</td>
			<td>K1621</td>
			<td>cDNA conversion kit</td>
		</tr>
		<tr>
			<td>2X SYBR Green Master Mix</td>
			<td>Roche</td>
			<td>4707516001</td>
			<td>qRT-PCR Master Mix</td>
		</tr>
		<tr>
			<td>FACS buffer</td>
			<td>Fisher Scientific</td>
			<td>50-105-9042</td>
			<td></td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Sigma Aldrich</td>
			<td>288306</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium acetate</td>
			<td>Sigma Aldrich</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Zebrafish Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tuebingen</td>
			<td>ZIRC</td>
			<td>ZL57</td>
			<td></td>
		</tr>
		<tr>
			<td>ins2a:mCherry</td>
			<td>ZIRC</td>
			<td>ZL1483</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40 micron cell strainer</td>
			<td>Sigma</td>
			<td>CLS431750</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tube</td>
			<td>BD Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Sigma</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina HiSeq</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480</td>
			<td>Roche</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mating tanks 1.0L Crossing Tank Set</td>
			<td>Aquaneering</td>
			<td>ZHCT100</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tube 5 mL polypropylene tube</td>
			<td>BD Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Consensus Path DB</td>
			<td>http://cpdb.molgen.mpg.de/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GO Enrichment Analysis</td>
			<td colspan="2">http://geneontology.org/page/go-enrichment-analysis</td>
			<td></td>
		</tr>
	</tbody>
</table>

sample preparation and analysis of rnaseq-based gene expression data from zebrafish

分析全球基因表达变化是一个有价值的工具, 以确定新的途径的基础观察表型。斑马鱼是一个很好的模型, 快速评估整个转录从整个动物或单个细胞的数量, 由于 RNA 的容易分离, 从大量的动物。本文介绍了利用 RNA 测序 (RNASeq) 对斑马鱼胚胎进行全球基因表达分析的协议。我们描述的是从整个胚胎或细胞群中获得的 RNA 的制备方法。我们还描述了一种分析 RNASeq 数据的方法, 用以识别全球基因表达数据集的富集通路和基因本体 (GO) 术语。最后, 我们提供了一个协议, 以验证基因表达变化使用定量逆转录酶 pcr (qRT pcr)。这些协议可用于比较分析的控制和实验组斑马鱼, 以确定新的基因表达变化, 并提供分子洞察的表型的兴趣。

差异表达基因的分类:
为了鉴别斑马鱼 Alström 综合征和 Bardet-Biedl 综合征 (BBS) 模型幼虫期的差异表达基因, 我们以alms1或bbs1的记录为目标, 将先前经过验证的拼接阻断 MOs 注入野生型斑马鱼胚胎16,17。在5天后受精 (dpf), 两个 RNA 复制从每个条件提取, 以及胚胎注射控制钼, 如?...

Watch this Scientific Journal Video about Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish at JoVE.com

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

该协议提出了一种从斑马鱼胚胎, 幼虫, 或排序细胞的整体转录分析的方法。我们包括 RNA 的分离, RNASeq 数据的通路分析, 和 qRT pcr 验证基因表达的变化。

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Research

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Genetics

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Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

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Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

通过两个互补细胞同步方案研究细胞周期调控的基因表达

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

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Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

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The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

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Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

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Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

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Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

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We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

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Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...