Name: sample preparation and analysis of rnaseq-based gene expression data from zebrafish
Uploaded: 2017-10-27T12:30:00.000+00:00
Duration: 11 min 42 s
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 insulin2a:mCherry 荧光报告株可以分析胰腺和 #946;-细胞. </li>
		<li> 将鱼转移到配有新鲜系统水的交配罐中, 并在第二天早上灯亮后立即移除分隔条. </li>
		<li> 允许鱼自然交配, 直到底部罐中的胚胎被观察到。以30分钟的间隔收集胚胎, 直到收集到所需的数量。将每个收集的时间点存储在28.5 和 #176 的胚胎培养基中的不同培养皿中; C. </li>
		<li> 在实验培养基中进行遗传物质或放置的显微注射 6 , 如果需要, 并培养新鲜的汉克和 #39 的胚胎培养基 8 在10厘米的 Petri 皿中28.5 和 #176; 注: 对于吗 (MO) 注入或突变动物的基因表达分析, 请注意, 每个操作都可能对基因表达产生附带影响。突变体可以在转录水平表现出基因补偿, 而 MO-based 基因的目标是 9 . </li>
	</ol> </li> <li> 阶段胚胎 <ol> <li> 在 50 和 #8211 的组中培养胚胎; 每10厘米培育皿中有75胚 以促进所有胚胎的一致发育时间. </li>
		<li> 在卵、外包和节阶段使用解剖显微镜监测发育形态学, 以确保发育进程 10 . 
		注: 去除任何垂死或畸形的胚胎, 以防止在盘中发育迟缓. </li>
		<li> 根据发育年龄分离胚胎。测量胚胎年龄使用节数字在分割以后 (post-gastrulation, 10.33 h 岗位受精 (hpf)) 直到大约 24 hpf。在 24 hpf 后, 用总体长来分期胚胎和幼虫. 
		注意: 节是在胚胎的背侧出现的 v 字形的胚层组织. </li>
		<li> 将已排序的胚胎放入28.5 和 #176; C 孵化器, 使发展达到预期的年龄. </li>
	</ol> </li> </ol> 2。单细胞离解: 整个胚胎和排序的细胞数量 <ol> <li> 整个胚胎分离 <ol> <li> 在预期的阶段安乐斑马鱼胚胎, 将培养皿放在冰上5-15 分钟, 直到没有观察到运动.</li>
		<li> 将20胚胎池转移到标记为1.5 毫升的离心管上。从离心管中取出多余的胚胎介质. </li>
		<li> 添加200和 #181; L 溶解试剂 (参见 材料表 ) 到含有胚胎的试管中。用杵质机械裂解试剂中的胚。添加800和 #181; L 溶解试剂, 使总体积为1毫升。存储在-80 和 #176; C 直到 RNA 提取. </li>
	</ol> </li> <li> 流排序的单元隔离 11 <ol> <li> 将胚胎从培养皿转移到1.5 毫升的离心管中, 并尽可能移除胚胎培养基. 
		注意: 根据胚胎年龄, 在这一步也可能需要机械离解。与胚胎和 #62; 48 hpf, 我们建议使用杵来破坏胚胎的完整性, 然后再继续. </li>
		<li> 孵育1毫升的离解缓冲 1 (见 材料表 ) 在1.5 毫升离心管的胚胎。室温孵育15-30 分钟。研制的解决方案, 轻轻地移向上和向下使用 P1000 提示混合每3-4 分钟在孵化步骤。不要涡流. </li>
		<li> 用离心法收集胚胎3分钟, 在 300 x 克和清除上清液。在1毫升离解缓冲2中重新暂停胚胎 (请参见 材料表 )。在室温下孵育15-30 分钟, 定期吸管研磨. </li>
		<li> 在显微镜下, 通过稀释1-2 和 #181 对荧光活化的细胞分类器中的悬浮液进行消化. 
		注意: 当被检查的分显示单细胞的细胞簇和胚胎组织的片断时, 完全消化已经发生. </li>
		<li> 以 300 x g 的离心法收集细胞, 5 分钟去除上清液。将细胞重新悬浮到1毫升的缓冲区, 重力过滤器通过40和 #181; m 细胞滤网从样品中去除未消化的组织. </li>
		<li> 使用例计算单元格, 并将其与资产管制的缓冲区稀释到大约 1x 10 6 在一个资产管制管中的单元/mL. 
		注: 对于每个胚胎 (少于5% 的总) 的细胞类型, 如胰腺和 #946;-细胞, 使用至少1000阶段匹配的胚胎. </li>
		<li> 为资产管制系统分类设备提供含有1毫升悬浮液样品的资产管制系统, 以及只含有裂解试剂或外地资产管制系统缓冲的资产管制系统管。门的流量分拣机只收集单一的细胞, 表达的荧光记者. </li>
		<li> 在收集到所需的单元格编号之前执行资产管制流程排序. 
		注意: 要执行 RNASeq, 有一个最小的总 RNA 需要。我们发现 3,000-5 000 细胞足以分离高质量高浓度的 RNA. </li>
		<li> 将收集的细胞储存在冰上, 并立即进行 RNA 制备. </li>
	</ol> </li> </ol> 3。rna 准备 <ol> <li> 提取 rna <ol> <li> 在室温下孵育所收集的样品 (从步骤 2) 和裂解试剂5分钟. </li>
		<li> 为每1.0 毫升的裂解试剂添加0.2 毫升氯仿, 并在十五年代用手倒置试管. 室温下孵育2-3 分钟。离心样品在 1.2万 x g 为15分钟在4和 #176; C. </li>
		<li> 将分离的, 水相转移到一个新鲜的管, 并添加0.5 毫升的异丙醇为每1.0 毫升的裂解试剂使用。室温孵育10分钟。离心样品在 1.2万 x g 为10分钟在4和 #176; C. </li>
		<li> 用75% 乙醇洗涤, 并将样品在 7500 x g 上离心5分钟, 在4和 #176; c. 完全去除上清, 使空气在室温下干燥。在15-30 和 #181 中重新悬浮 RNA; 焦二乙基 (DEPC)-处理过的水. </li>
	</ol> </li> <li> 纯化高质量的 rna <ol> <li> 将 RNA 悬浮与1/10 卷3M 醋酸钠 (pH 5.5) 和1的异丙醇量结合起来。在室温下孵育20分钟, 将样品在 12500 x g 离心10分钟, 在4和 #176; C. </li>
		<li> 在 DEPC 处理过的水中用 ice-cold 70% 乙醇清洗颗粒, 并将样品在 1万 x g 上离心5分钟, 在4和 #176; 重复一次洗涤步骤. </li>
		<li> 清除上清, 允许在室温下干燥。重新悬浮在 DEPC 处理的水中. </li>
		<li> 使用吸收光谱仪测定提取的 RNA 浓度 (ng/和 #181; L) 和纯度 (260/230 比)。在继续 RNASeq 之前, 请确保260/230 值为 ~ 2.0。存储在-80 和 #176; C 直到我们e (最多6月). </li>
		<li> 将 RNA 样本发送给供应商或核心以进行 RNASeq 和基因表达变化分析, 这是基于顺序读取的量化。从供应商返回的结果通常提供的基因列表显示, 在样本之间的成绩单读取明显的折叠变化. 
		注: 如果上述方法产生质量较差的 RNA, 则可以使用列。rna 样本的数量、浓度和 rna 完整性数字应与供应商或核心确认。供应商或核心也通常会评估. </li>
	</ol> </li> </ol> 4。路径和 GO 术语分析 注意: 在核心或供应商提供的 RNASeq 后, 请参阅 图 1 , 以了解基因表达分析的代表性输出. 
<ol> <li> 对差异表达的基因排序 <ol> <li> 在电子表格管理软件中比较单个实验条件与控制 <ol> <li> 打开数据 (结果) (请参见 材料表).</li>
			<li> 选择 #39 旁边的下拉箭头; 排序和 #39; 按钮并选择和 #34; 自定义排序和 #34; 在包含实验与控制条件中的差异表达基因的电子表格中. </li>
			<li> 选择 #39 下的框; 列和 #39; 在弹出的窗口中, 选择按和 #39 排序; 利物浦和 #39; 专栏。确保和 #39; 价值和 #39; 在和 #39 中选择; 排序 #39; 列。选择以排序从和 #39; 最大到最小和 #39; 在和 #39; 命令和 #39; 列并单击和 #34; 确定和 #34;. 
			注: 这将排序差异表达的基因, 使那些增加表达 (正面利物浦) 将出现在列表的顶部, 而那些减少表达 (负利物浦) 将出现在列表的底部. </li>
		</ol> </li> <li> 将多个实验条件与单个控件进行比较, 如下所示。
		<ol> <li> 在实验1与控制电子表格中选择空列中的第一个单元格, 以确定在两个实验条件中发现的差异表达基因。在单元格中键入以下公式: <img alt="Equation" src="/files/ftp_upload/56187/56187eq1.jpg"/> 注意: 此公式是 IF、ISERROR 和匹配函数的组合。(i) 比赛功能, 比赛 (Experimental2_vs_Control!A:A,0), 将查找 A1 (ENSEMBL ID) 中的值, 该列 (a:) 的电子表格名为 Experimental2_vs_Control (Experimental2_vs_Control!)。#34; 0 和 #34; 指示查找精确匹配, 如果找到精确匹配, 则函数将返回值 #34; 1 和 #34; 如果未找到匹配项, 则函数将返回错误和 #34; N/a 和 #34;。(ii) 比赛功能的结果, 然后输入到 ISERROR 函数, ISERROR (匹配 (A1, Experimental2_vs_Control!A:A,0)), 如果输入是和 #34; 1 和 #34; 表明匹配被发现, 它将返回和 #34; 假和 #34; 如果输入是和 #34; N/a 和 #34; 表示未找到匹配, 则返回和 #34; 真和 #34;。(三) #34; 真实与 #34; 或 #34; 虚假和 #34; 结果然后输入 if 函数, 如果 (ISERROR (Experimental2_vs_Control!A:A,0), #34; #34 #34;D 重复 #34;) 在哪里, 如果输入是和 #34; 真实和 #34; 表示未找到匹配项, 则将返回第一组引号 (和 #34; #34;) 为空, 因此单元格将为空。如果输入 #34; FALSE 和 #34; 表示匹配, 则将返回第二组引号 (和 #34;D 重复和 #34;) 之间的值, 并且单元格会说和 #34;D 重复 #34;. </li>
			<li> 按 #34; 输入和 #34; 或 #34; 返回和 #34; 运行等式。选择包含公式的单元格, 然后单击单元格右下角的框。保持鼠标单击并将所选区域一直拖到列中, 直到最后一个 #39; 功能 ID 和 #39;, 将公式复制到列中的每个单元格中. </li>
			<li> 选择和 #34; 自定义排序和 #34; 再次, 通过单击 #34; + #34; 左下角的图标, 添加第二级排序。在第一级, #34; 排序依据和 #34;, 在列选择和 #34;D 重复和 #34;, 根据选择和 #39 排序; 值和 #39;, 根据顺序选择和 #39; Z 到 A 和 #39;。在第二级, #34; 然后通过和 #34;, 在列选择和 #39; 利物浦和 #39; 根据选择和 #39; 价值和 #39; 根据顺序选择和 #39; 最大到最小和 #39; 选择和 #34; 好 #34;. 
			注意: 这将根据表达变化的方向性将基因列表分类为4组。重要的是要检查在两个实验条件中发现的基因, 以确保表达的变化是在同一方向的两个或相反的方向. </li>
			<li> 在继续之前从基因符号列中删除任何括号, 否则在数据库中将找不到结果。选择包含基因符号的整列。选择和 #34; 编辑和 #34; 然后, #34; 替换和 #34; 在下拉 #39; 文件和 #39; 菜单。类型和 #34;(*) 和 #34; 在和 #34; 查找: 和 #34; "替换" 窗口的栏, 然后离开和 #34; 替换为: 和 #34; 栏为空。选择和 #39; 替换所有 #39; 删除括号中的所有实例. 
			注: * 表示任意数量的字符, 通过在两个圆括号之间放置此字符, 程序将提示您在突出显示的单元格中查找任何实例, 并且任何括号实例都将被替换为无, 从而删除它们. </li>
		</ol> </li> </ol> </li> <li> 确定富集路径 <ol> <li> 复制要确定剪贴板的丰富路径的集合的基因符号。转到 ConsensusPathDB 12 。选择和 #34; 基因组分析和 #34; 在网页的左栏, 其次是和 #34; 过度表征分析和 #34;. </li>
		<li> 将 #34 中的基因列表粘贴;P 浪费基因/蛋白质标识符和 #34 的列表; 框。在 #34 中选择基因符号; 基因/蛋白质标识符类型和 #34; 框和单击 #34;P roceed 和 #34;。根据路径数据库和 #34 的定义, 选择;P athways 旁边的框和 #34; 在 "Pathway-based 集" 部分下. 
		注意: 将显示许多用于分析的选项, 包括要搜索的可用数据库的列表。我们建议取消所有数据库, 除了 Kegg 和 Reactome 数据库, 因为它们是最建立和全面的。还可以根据需要调整与输入列表和 p 值截止设置的最小重叠。我们建议将这些设置保留在默认的2基因最小重叠和 0.01 p 值截止处. </li>
		<li> 选择和 #34; 查找丰富集和 #34; 获取输入列表中包含基因的路径列表. 
		注: 输出将以表格的形式, 包括每个通路的名称, 其次是和 #34; 设置大小和 #34; (指明通路中总基因的数量); #34; 候选人包含和 #34; (表明输入列表中的基因数量在该通路中), 以及 p 值和 q 值 (罗斯福), 和数据库的路径被确定. </li>
	如上所述, </ol> </li> <li> 生成路径网络 <ol> <li> 确定丰富的路径. </li>
		<li> 选择所有富集路径以在通路网络中可视化, 通过选择要可视化的路径名称旁边的每个框, 或单击和 #34; 全部和 #34; 下 #34; 选择和 #34; 在选择框上方的列标题中, 选择和 #34;可视化所选集和 #34;. 
		注意: 我们建议, 如果有少于30的所有路径的可视化;如果有大于30的路径, 我们建议选择前30最丰富的路径 (那些含有最大数量的基因从输入列表). </li>
		<li> 调整和 #34; 相对重叠和 #34; 和 #34; 共享候选和 #34; 筛选器, 通过选择页面顶部中心的任一框并输入渴求百分比相对重叠或共享候选的数量, 要更加严格, 以便使路径网络中的更贴切的重叠更清晰, 并选择和 #34; 应用和 #34;. 
		注意: 我们建议最小的相对重叠 0.2, 表明20% 基因重叠之间的2通路, 以连接他们, 至少2共享候选人。通过单击页面左上角的图形图例可以看到图形图例. </li>
	</ol> </li> <li> 确定丰富的 GO 术语 <ol> <li> 复制该组的基因符号, 其中一个希望将丰富的基因本体确定为剪贴板。转到 #39; go 富集分析工具和 #39; 在基因本体论联合体中 13 。将基因符号列表粘贴在页面左侧的框中, #34; 您的基因 id 在这里和 #8230; #34; </li> <li> 选择要使用的 GO 术语集, 在 "基因 id" 框下面列出: 生物过程、分子功能或细胞成分。选择 (推荐) 和 #39; 生物过程和 #39;选择和 #39; 斑马鱼和 #39; 在 GO 术语框下方, 单击并 #34; 提交和 #34; 
		注意: 我们建议使用默认的 p 值截断 0.05. </li>
	</ol> </li> </ol> 5。qRT pcr 验证方法 注: 在复制实验中, 应通过靶向 qRT pcr 验证 RNASeq 基因表达变化的个体基因. 
<ol> <li> cDNA 合成 <ol> <li> 使用3.1 节中描述的方法从胚胎中提取 RNA。RNA 提取. </li>
		<li> 转换1和 #181; 使用 cdna 转换试剂盒将 RNA 转化为 cdna (请参阅 材料表 )。结合 RNA、dNTPs 和逆转录酶。根据制造商和 #39 的规格执行 thermocycler 反应. </li>
		<li> 在 DEPC 处理的水中稀释1:3 的 cDNA。存储在4和 #176; C 最多1-2 月. </li>
	</ol> </li> <li> qRT-pcr 验证 <ol> <li> 选择由 RNA 测序结果 qRT pcr 验证的基因。在表达中发现具有高折叠变化的基因。确定这些基因中哪一个也含有高的读数, 因为这表明了丰富的表达. </li>
		<li> 选择10-12 目标从高折叠变化, 高读取计数列表的基因。设计引物, 以放大跨越至少1内含子外显子边界的目标基因. </li>
		<li> 将基因或目标区域输入 qPCR 入门设计站点 14 。选择 #34;d 设计 2 qPCR 底漆和 #34; 并提示网站创建入门集. 
		注意: 一定要包括内部控制底漆, 例如 和 #946;-肌动蛋白 , 以规范样本之间的比较。#946;-肌动蛋白底漆为 F: 5 和 #39; TCGAGCTGTCTTCCCATCCA-3 和 #39; R: 5 和 #39;-TCACCAACGTAGCTGTCTTTCTG-3 和 #39;. </li>
		<li> 将 cDNA、前引物、反转底漆和聚合酶结合。进行 qRT PCR 扩增, 以确定基因的相对表达 15 . </li>
		<li> 通过相对量化测定来分析感兴趣基因的相对 mRNA 表达 15 . </li>
		<li> 将 qRT PCR 与 RNASeq 结果进行比较, 以确保差异表达式的方向性一致. </li>
	</ol>
	</li>
</ol>

<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>

作者没有什么可透露的。

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>&lt;strong&gt;Commercial Reagents&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Zebrafish Strains&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</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>http://geneontology.org/page/go-enrichment-analysis</td><td></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 ...

sample-preparation-analysis-rnaseq-based-gene-expression-data-from

Research

JoVE Journal

Genetics

10.8K 次观看.  University of Maryland School of Medicine. 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。

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

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

大规模的斑马鱼胚胎的心脏解剖的转录分析

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

科研

发育生物学

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

遗传学

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

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

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

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 ...

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 ...

<meta charset="utf8"></head><body>使用 CRISPR/Cas9 敲入在斑马鱼幼虫中进行荧光蛋白标记

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

<meta charset="utf8"></head><body>荧光标记以可视化斑马鱼中的低表达蛋白质

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

荧光标记以可视化斑马鱼中的低表达蛋白质

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

处理斑马鱼幼虫的肠道细胞以进行单细胞 RNA 测序

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

作者聚焦:分离和分析斑马鱼幼虫的肠道细胞以研究胃肠道发育的转录组学方面 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

摘要

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此视频中的章节

大规模的斑马鱼胚胎的心脏解剖的转录分析

使用斑马鱼作为体内模型系统分析 DNA 复制定时

分离和斑马鱼胚胎单细胞的表征

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

全球基因表达分析，使用斑马鱼的寡核苷酸芯片平台

荧光标记以可视化斑马鱼中的低表达蛋白质

荧光标记以可视化斑马鱼中的低表达蛋白质

荧光标记以可视化斑马鱼中的低表达蛋白质

为早期小鼠胚胎测序准备小型RNA库

从斑马鱼幼虫中分离肠道进行单细胞RNA测序