Name: an experimental and bioinformatics protocol for rna-seq analyses of photoperiodic diapause in the asian tiger mosquito, aedes albopictus
Uploaded: 2014-11-30T15:00:00.000+00:00
Duration: 12 min 10 s
Description: Watch this Scientific Journal Video about An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus at JoVE.com

This work was supported by the National Institutes of Health grant 5R21AI081041-02 and Georgetown University.

两个A. 1.苗种培育白纹伊蚊组以共创成长<ol><li>设置两个光周期柜带有可编程照明在21℃为最佳滞育表达16和大约80％的相对湿度。 <ol><li>计划一个机柜为16L:8D光:暗周期（非滞育诱导LD光周期）。设置第二个柜的8L:16D光:暗周期（滞育诱导）13。 </li><li>程序"灯"在同一时间在两个机柜来同步光周期之间的昼夜时间。 </li></ol></li><li>计算来执行实验所需的蛋的数量。瞄准每笼300-500鸡蛋。至少有三个复制滞笼和三个复制所需的RNA生成非滞育笼。这总计为长约每个实验1,800-3,000鸡蛋。 <ol><li>孵化的鸡蛋鸡蛋浸入到的论文约 500ml的去离子H 2 O.的</li><li>添加&lt;EM&gt; CA。1毫升食物制备由地面狗食和盐水虾如前所述24。覆盖容器目，并保持网格代替用橡皮筋。 </li><li>放置在LD光照柜约 24小时。 </li></ol></li><li>转印孵出幼虫至10×10×2cm的培养皿填充有大约 90毫升去离子H 2 O. <ol><li>维持每盘约 30幼虫。幼虫转移到清洁的菜肴每48-72小时，例如每周一，周三和周五（MWF）24。 </li><li>饲料约 1毫升食物浆每个​​MWF包括地面狗粮和盐水虾去离子水如前面所述24。 </li></ol></li><li>设立三到四个成年笼子每个光照治疗，其中每个笼包括生物学重复。 <ol><li>从9.5 L桶相对两侧，切出一个10×14厘米的洞，并且另一个孔15厘米直径的。有限公司版本1.00先用网。切的矫形放养约一英尺长度，和胶水围绕另一孔的内侧一端。 </li><li>削减库存关闭，结关的足端 - 开放的，只有当进入笼子的内部需要。对于笼盖，切出的所有的内部，只留下边，并与网孔24代替内部塑料。 </li><li>注意光照，复制相关的实验上笼的一侧永久性标记号，笼起始日期等信息。 </li></ol></li><li>行成人笼用湿滤纸的底部。挫伤滤纸用足够的去离子H 2 O来增加局部湿度在笼中，但要避免积水，这可以刺激产卵在滤纸24上 。每天检查滤纸干燥，再湿必要时。 </li><li>以产生足够的鸡蛋的RNA库，包括至少100位女性/ 9.5 L CAGE，没有每笼500多蚊子。 </li><li>收集蛹MWF并将其放置在一个小杯子干净的H 2澳在不超过50蛹每25毫升H 2 O.密度蛹杯转移到一个成年人的笼子。笼子放置在相应的光照柜- A.白纹伊蚊蛹的感光15。 </li><li>确保日常使得H 2 O的杯子是干净，清晰，并去除死蛹，因为积聚死蛹会导致大量死亡。除去H 2Ø杯毕竟蛹出现。 </li><li>将有机葡萄干上笼提供糖羽化成虫的顶网。监测葡萄干，并改变他们每隔3-5天，以防止霉菌的积累。 </li></ol> 2.维护成人为允许交配和产蛋<ol><li>通过用湿滤纸衬笼底部保持笼子在高湿度（约80％），并提供访问非发霉葡萄干，如上所述（Secti附件1.5和1.9）。 </li></ol> 3.吸血<ol><li>准备血液供给雌性羽化后二至六天之间，以确保女性已经暴露于至少八个无歧义短天前开始产卵的近100％的滞育卵14。 </li><li>准备Hemotek膜进给系统。堵塞进料装置进入电源。用调整螺钉调整每个单元的温度升高到37℃。使用电子温度计和探头校准期间测量所述馈送单元的温度。 </li><li>准备一顿水库。拉伸的胶原膜馈送一个正方形在膳食储存器的孔，并与O形环固定。小心地将去除皱纹的角落;修剪多余的膜用剪刀。 注:胶原蛋白膜可能不适用于所有蚊种工作顺利，可能需要尝试几种类型，以找到最佳的膜，如果工作有品种较其他A.白纹伊蚊 。封口膜可以很好地淡色库蚊 。 <ol><li>如果血液被冷冻贮存，在室温下解冻，至少1小时，使用前。 </li><li>持这样的膜朝下的库，不支持的，以及该填充端口被朝上。使用移液管或注射器填充储与大约3毫升鸡全血具有柠檬酸钠作为抗凝血剂。轴封采用塑料塞填充端口。 </li></ol></li><li>通过拧到它上的馈线的底部传热板的螺栓连接的制备储到馈线。倒置馈线并将其放置在顶笼，膜面朝下，使蚊子可以通过笼的网孔送入。保持馈线上笼约45分钟，以最大限度地喂养。 </li></ol> 4.刺激产卵<ol><li>四，五天后血粉，装备每笼有色深色50毫升Ç向上排列着未漂白种子发芽纸（卵纸）或纹理的非漂白纸毛巾，并与去离子水9中途补。如果超过250蚊子是关在笼子里，用两个杯子。 注:对于小型笼或单女小瓶，在产卵箱"干草输液"可能会增加产卵由于微生物菌群25的气味。 </li></ol> 5.收集和储存鸡蛋<ol><li>在4-5天的血液喂养开始，因为五天后血液喂养产蛋量一般约达到高峰，然后逐渐消退，未来一周蛋收集。 </li><li>不同的实验用品蛋收集频率。对于一般用途，收集了MWF如期鸡蛋论文。从每个网箱中取出卵子的论文，换上新纸。将在培养皿中，并存储在SD光照内阁最近删除的文件，以避免鸡蛋储存的混杂影响。 </li><li>让鸡蛋论文重新<html>主要湿2天后产卵，以允许浆膜角质层的形成，从而增加了蛋脱水电阻26。 </li><li>大约48小时后收集，干燥蛋露天。干的纸张，这是跛行，微湿的触感，但没有这么潮湿的纸张不是H 2 O暗或刺激种蛋。 注:6.5"×4"文件大约需要3.5小时，晾干。要小心，不要过度干燥蛋的论文，因为这将导致鸡蛋干燥27。 </li><li>储备来自LD和SD光周期的额外的鸡蛋，评估滞育发病率和解释的光周期的影响（见测量滞育，第6条）。 </li><li>对于长期储存，保持蛋纸在21℃和大约80％的湿度在培养皿中。保持培养皿在特百惠的存储容器的水瓶来维持当地的湿度胚胎发育需要四到五天，在21°C。 </li></ol>ove_title"&gt; 6，测量滞育发病率<ol><li>使用额外预留胚胎（见刺激产卵，第4节）是7-20日龄量化滞育的响应。 </li><li>记录本上每个蛋的蛋的纸张的数量。 </li><li>刺激鸡蛋完全淹没个别蛋的论文中90毫升培养皿约80毫升去离子H 2 O.孵化加入约0.25毫升食物浆。 </li><li> 24小时后相符斜线第一龄幼虫的数目。放置在黑色表面培养皿形象化幼虫和放置一个光源上盘的一侧。幼虫将移动远离光源，允许个别幼虫的明确讯号。用吸管删除单个的幼虫，而计数，以防止个别讲述幼虫。 </li><li>地方鸡蛋论文在一个新的培养皿中，然后再干燥。再孵化鸡蛋后约1周，再吻合鸡蛋使用上述方法孵化。 </li><li>地方鸡蛋篇，其余ü<html>正孵化的鸡蛋在新90毫升培养皿约80ml漂白液28。确保蛋论文在漂白溶液完全淹没，并在通风橱中过夜离开，以避免漂白剂的气味。 注:漂白溶液可存放约1星期4℃，但应以其他方式进行的新鲜。 </li><li>使用光学显微镜作为漂白将清除绒毛膜和允许胚，未孵出的卵的可视化检查蛋。如果蛋是胚，卵将具有灰白色色与眼睛显示为两个小的黑点彼此相对的背侧。理货未阴影，鸡胚13的数量。 </li><li>确定滞育率用下列公式计算:％滞育=无。胚未孵化的蛋/（无，孵化蛋+没有。胚未孵化的蛋）×100 13。 </li></ol> 7. RNA提取鸡蛋/ pharate幼虫ontent"&gt;注:使用的Trizol在层流罩。 <ol><li>刷蚊卵含胚胎发育或pharate幼虫在不同的发育时间点从卵纸用驼毛刷玻璃磨床。在研磨Trizol法鸡蛋（每50-100 mg组织1毫升），直到完全粉碎。至少使用400的鸡蛋每个库，以产生足够的RNA。 <ol><li>或者，捕捉冷冻蛋在液氮中，并储存在-80℃下在微量离心管长达一个月磨削中的Trizol之前。 </li></ol></li><li>根据制造商的说明在Trizol试剂进行RNA提取，随后用异丙醇沉淀。 </li><li>治疗用核糖核酸酶净化解决方案或其他代理人的替补，以消除任何残余的核酸，以避免RNA降解。 <ol><li>把提取的RNA与DNA酶。根据制造商的说明，孵育RNA样品用DNase 30分钟，在37℃。使用1＃181;在一个50μl的反应升DNA酶长达10的RNA微克。增加DNA酶的量，如果有超过10个的RNA微克在一个反应​​。 </li><li>通过加入5微升悬浮DNA酶失活试剂灭活脱氧核糖核酸酶。孵育5分钟，在室温下，在孵育期间（平缓涡旋）混合三次。 </li><li>离心以10,000 xg离心1.5分钟。传送含有经处理的RNA样本到新管用于后续步骤中的上层清液。 </li></ol></li><li>评估总RNA样品用荧光的质量。送样品到适当的仪器完成这个任务，专业设施。该设施将执行片上的凝胶电泳，以确定RNA种类的样品中，通过荧光染料灌输在芯片可视的大小。结果将被返回作为电泳。 <ol><li>测定总RNA样品的存在或不存在的降解产物的完整性，由此证明存在Ò关于所得到的电泳图（ 图1B）的18S和5S核糖体RNA的峰F之间的峰。 </li></ol></li></ol> 8. RNA测序<ol><li>送的总RNA样品以足够高的质量（ 图1A）和数量（每个库通常&gt; 3微克），以一个商业测序中心建筑富集配对末端文库的mRNA和mRNA测序，下列标准协议。 </li><li>如果一个以上的车道被用于测序的单个实验中，分割各个库成两个车道进行测序，以占测序期间车道之间的技术差异。 </li></ol> 9. lllumina读清洗 注:图2总结了本协议的生物信息学部分。对于本协议的生物信息学段中使用的所有程序和资源的完整列表，请参阅表1。在此外，补充文件1包含的命令行的示例为下列每个生物信息的协议的步骤。 <ol><li>使用ssaha2 29（ 表1），以确定95％的同一性或更高的匹配，以在NCBI UniVec Core数据库（ 见表1），A。 白纹伊蚊 rRNA序列（GenBank中＃L22060.1），和测序适配器（以补充文件1提供了详细的命令行的例子）。通过调整提供的Perl脚本（参考文件2）拆下读对，使用Perl类似的脚本工具或火柴，例如。 </li><li>与SolexaQA封装30清洁残留读取（ 表1;补充文件1）:修剪的区域用PHRED得分当量小于20使用DynamicTrim.pl的默认设置。 <ol><li>卸下两个读取短于25个碱基，LengthSort.pl正向和反向同时读取。评价清洁FASTQ文件与FastQC（ 表1的质量</st荣&gt;） - 尤其是，验证每个碱基序列的质量和每个序列的质量分数均高于20。 </li></ol></li></ol> 10.数字归<ol><li>在清洁过读取用高棉工具31执行一个循环数字正常化（ 表1;补充文件1），具体而言normalize-by-median.py（使用K-聚体大小20，一个覆盖截止20，并且x = 1E10）。 </li><li>另外，如果一台机器具有高RAM可用（数百GB的），使用三位一体的normalize_by_kmer_coverage.pl脚本（ 表1）。 </li></ol> 11. 从头转录组大会<ol><li>获得访问计算机或计算机集群具有多达256个GB的RAM和24个CPU，取决于组件的尺寸。 </li><li>使用三一32（ 表1;补充文件1）组装的数字归读设置成重叠群。为了减少内存使用情况，使用--min_kmer_cov 2。 </li></ol> 12.大会评估<ol><li>从三位一体重叠群输出Assemblathon2项目33运行assemblathon_stats.pl。这个脚本执行相关的评估装配质量的基本计算，如一些支架，N50，装配组合物，更（ 表1;补充文件1）。 </li></ol> 13.注释的组装转录组<ol><li>执行与参考蛋白质组的组件的BLASTX（ 表1）;蚊子， 果蝇，冈比亚按蚊，库蚊和白纹伊蚊都适合引用。具体来说，格式化参考蛋白质FASTA文件爆炸，随后BLASTX（参考文件1）。 </li></ol> 14.地图读取大会使用RSEM 34（表1） <ol><li>创建一个谈话到基因图"文件，其中的第一列含有参照基因的ID，并且第二列中的重叠群的ID。在一个电子表格编辑器，交换从BLASTX输出的第一和第二列，而写这些列.txt文件。使用断行程序转换换行符导致.txt文件为Unix格式。 </li><li>创建从转录FASTA文件中使用RSEM-准备参考脚本，在RSEM包（补充文件1）提供的参考数据集。 </li><li>分别计算出的表情值用于使用RSEM-计算表达命令，在RSEM包（参考文件1）提供的每个库。作为读出，则使用从读清洗步骤（步骤9.2）得到的配对FASTQ文件。 <ol><li>如果从生物复制的RNA被分裂成两个车道进行测序，包括在表达式计算两个FASTQ文件来产生一个单一的文件。 </li></ol></li><li>从每个文库中表达的结果转换成由另一个p容易地加工成矩阵使用提供的脚本RSEM生成数据矩阵，在RSEM包（参考文件1）提供rograms。 </li></ol> 15.差异表达分析<ol><li>安装R和轧边机（ 表1）。 </li><li>使用read.delim加载步骤14.3（参考文件1）RSEM结果。如果需要的话，圆的计数为最接近的整数。 注:磨边机指南推荐的数据集限制为基因的高表达足以检测的意义。 </li><li>要格式化的磨边机的数据，生成所加载数据文件（补充文件1）DGEList对象。然后，用标准化规范化TMM（参考文件1）中的数据。估计数据（参考文件1）共同和tagwise分散。 </li><li>找出差异表达的基因与的Benjamini-霍赫贝格校正p值&lt;0.05（参考文件1）。积数倍数变化与丰度（参考文件1）的分布。 </li></ol>

<ol>
	<li>Bilyk, K. T., Cheng, C. H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+of+gene+expression+in+extreme+cold+-+reference+transcriptome+for+the+high-Antarctic+cryopelagic+notothenioid+fish+Pagothenia+borchgrevinki.">Model of gene expression in extreme cold - reference transcriptome for the high-Antarctic cryopelagic notothenioid fish Pagothenia borchgrevinki.</a> BMC Genomics. 14, 634 (2013).</li><li>Chapman, M. A., Hiscock, S. J., Filatov, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+divergence+during+speciation+driven+by+adaptation+to+altitude.">Genomic divergence during speciation driven by adaptation to altitude.</a> Mol. Biol. Evol. 30, 2553-2567 (2013).</li><li>Schwarz, D., 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=Sympatric+ecological+speciation+meets+pyrosequencing:+sampling+the+transcriptome+of+the+apple+maggot+Rhagoletis+pomonella.">Sympatric ecological speciation meets pyrosequencing: sampling the transcriptome of the apple maggot Rhagoletis pomonella.</a> BMC Genomics. 10, 633 (2009).</li><li>Barshis, D. J., 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=Genomic+basis+for+coral+resilience+to+climate+change.">Genomic basis for coral resilience to climate change.</a> P. Natl Acad. Sci. USA. 110, 1387-1392 (2013).</li><li>Urbanski, J. M., Aruda, A., Armbruster, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptional+element+of+the+diapause+program+in+the+Asian+tiger+mosquito,+Aedes+albopictus,+identified+by+suppressive+subtractive+hybridization.">A transcriptional element of the diapause program in the Asian tiger mosquito, Aedes albopictus, identified by suppressive subtractive hybridization.</a> J. Insect Physiol. 56, 1147-1154 (2010).</li><li>Huang, Y. H., 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=The+duck+genome+and+transcriptome+provide+insight+into+an+avian+influenza+virus+reservoir+species.">The duck genome and transcriptome provide insight into an avian influenza virus reservoir species.</a> Nat. Genet. 45, 776-783 (2013).</li><li>Sessions, O. M., 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=Host+cell+transcriptome+profile+during+wild-type+and+attenuated+dengue+virus+infection.">Host cell transcriptome profile during wild-type and attenuated dengue virus infection.</a> PLoS Negl. Trop. Dis. 7 (3), 2107 (2013).</li><li>Poelchau, M. F., Reynolds, J. A., Elsik, C. G., Denlinger, D. L., Armbruster, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+sequencing+reveals+complex+mechanisms+of+diapause+preparation+in+the+invasive+mosquito,+Aedes+albopictus.">Deep sequencing reveals complex mechanisms of diapause preparation in the invasive mosquito, Aedes albopictus.</a> P. R. Soc B. 280, (2013).</li><li>Poelchau, M. F., Reynolds, J. A., Elsik, C. G., Denlinger, D. L., Armbruster, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+sequencing+as+a+platform+to+elucidate+molecular+components+of+the+diapause+response+in+Aedes+albopictus.">Transcriptome sequencing as a platform to elucidate molecular components of the diapause response in Aedes albopictus.</a> Physiol. Entomol. 38, 173-181 (2013).</li><li>Poelchau, M. F., Reynolds, J. A., Denlinger, D. L., Elsik, C. G., Armbruster, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+de+novo+transcriptome+of+the+Asian+tiger+mosquito,+Aedes+albopictus,+to+identify+candidate+transcripts+for+diapause+preparation.">A de novo transcriptome of the Asian tiger mosquito, Aedes albopictus, to identify candidate transcripts for diapause preparation.</a> BMC Genomics. 12, 619 (2011).</li><li>Benedict, M. Q., Levine, R. S., Hawley, W. A., Lounibos, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spread+of+the+tiger:+Global+risk+of+invasion+by+the+mosquito+Aedes+albopictus.">Spread of the tiger: Global risk of invasion by the mosquito Aedes albopictus.</a> Vector-Borne Zoonot. 7, 76-85 (2007).</li><li>Lounibos, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasions+by+insect+vectors+of+human+disease.">Invasions by insect vectors of human disease.</a> Annu. Rev. Entomol. 47, 233-266 (2002).</li><li>Urbanski, J. M., 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=Rapid+adaptive+evolution+of+photoperiodic+response+during+invasion+and+range+expansion+across+a+climatic+gradient.">Rapid adaptive evolution of photoperiodic response during invasion and range expansion across a climatic gradient.</a> Am. Nat. 179, 490-500 (2012).</li><li>Lounibos, L. P., Escher, R. L., Lourenco-de-Oliveria, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+evolution+of+photoperiodic+diapause+in+temperate+and+tropical+invasive+populations+of+Aedes+albopictus+(Diptera+Culicidae).">Asymmetric evolution of photoperiodic diapause in temperate and tropical invasive populations of Aedes albopictus (Diptera Culicidae).</a> Ann. Entomol. Soc. Am. 96, 512-518 (2003).</li><li>Mori, A., Oda, T., Wada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Studies on the egg diapause and overwintering of Aedes albopictus in Nagasaki. Trop. Med. 23, 79-90 (1981).</li><li>Pumpuni, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Factors influencing photoperiodic control of egg diapause in Aedes albopictus [dissertation]. , (1989).</li><li>Wang, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+influence+of+photoperiod+on+egg+diapause+in+Aedes+albopictus+Skuse.">Observations on the influence of photoperiod on egg diapause in Aedes albopictus Skuse.</a> Acta Entomol. Sinica. 15, 75-77 (1966).</li><li>Sota, T., Mogi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival-time+and+resistance+to+desiccation+of+diapause+and+non-diapause+eggs+of+temperate+Aedes+(Stegomyia)+mosquitoes.">Survival-time and resistance to desiccation of diapause and non-diapause eggs of temperate Aedes (Stegomyia) mosquitoes.</a> Entomol. Exp. Appl. 63, 155-161 (1992).</li><li>Reynolds, J. A., Poelchau, M. F., Rahman, Z., Armbruster, P. A., Denlinger, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcript+profiling+reveals+mechanisms+for+lipid+conservation+during+diapause+in+the+mosquito,+Aedes+albopictus.">Transcript profiling reveals mechanisms for lipid conservation during diapause in the mosquito, Aedes albopictus.</a> J. Insect Physiol. 58, 966-973 (2012).</li><li>Andrewartha, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diapause+in+relation+to+the+ecology+of+insects.">Diapause in relation to the ecology of insects.</a> Biol. Rev. 27, 50-107 (1952).</li><li>Danks, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+Dormancy:+An+Ecological+Perspective.">Insect Dormancy: An Ecological Perspective.</a> Biological Survey of Canada (Terrestrial Arthropods). , (1987).</li><li>Denlinger, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+diapause.">Regulation of diapause.</a> Ann. Rev. Entomol. 47, 93-122 (2002).</li><li>Rev Entomol, A. n. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 59, 93-122 (2014).</li><li>Armbruster, P. A., Conn, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geographic+variation+of+larval+growth+in+North+American+Aedes+albopictus+(Diptera).">Geographic variation of larval growth in North American Aedes albopictus (Diptera).</a> Culicidae). Ann. Entomol. Soc. Am. 99, 1234-1243 (2006).</li><li>Reiter, P., Amador, M. A., Colon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+the+CDC+ovitrap+with+hay+infusions+for+daily+monitoring+of+Aedes+aegypti+populations.">Enhancement of the CDC ovitrap with hay infusions for daily monitoring of Aedes aegypti populations.</a> J. Am. Mosquito Contr. Association. 7 (1), 52 (1991).</li><li>Rezende, G. L., 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=Embryonic+desiccation+resistance+in+Aedes+aegypti:+presumptive+role+of+the+chitinized+serosal+cuticle.">Embryonic desiccation resistance in Aedes aegypti: presumptive role of the chitinized serosal cuticle.</a> BMC Dev. Biol. 8, 182 (2008).</li><li>Munstermann, L., Crampton, J., Beard, C., C, L. o. u. i. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Care+and+maintenance+of+Aedes+mosquito+colonies.">Care and maintenance of Aedes mosquito colonies.</a> The Molecular Biology of Insect Disease Vectors. , 13-20 (1997).</li><li>Trpis, 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+new+bleaching+and+decalcifying+method+for+general+use+in+zoology.">A new bleaching and decalcifying method for general use in zoology.</a> Can. J. Zoolog. 48, 892-893 (1970).</li><li>Ning, Z. M., Cox, A. J., Mullikin, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SSAHA:+A+fast+search+method+for+large+DNA+databases.">SSAHA: A fast search method for large DNA databases.</a> Genome Res. 11 (10), 1725-1729 (2001).</li><li>Cox, M. P., Peterson, D. A., Biggs, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SolexaQA:+At-a-glance+quality+assessment+of+Illumina+second-generation+sequencing+data.">SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data.</a> BMC Bioinformatics. 11, 485 (2010).</li><li>Grabherr, M. G., 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=Full-length+transcriptome+assembly+from+RNA-Seq+data+without+a+reference+genome.">Full-length transcriptome assembly from RNA-Seq data without a reference genome.</a> Nat. Biotechnol. 29 (7), 644-652 (2011).</li><li>Bradnam, K. R., 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=Assemblathon+2:+evaluating+de+novo+methods+of+genome+assembly+in+three+vertebrate+species.">Assemblathon 2: evaluating de novo methods of genome assembly in three vertebrate species.</a> GigaScience. 2, 10 (2013).</li><li>Li, B., Dewey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RSEM:+accurate+transcript+quantification+from+RNA-Seq+data+with+or+without+a+reference+genome.">RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.</a> BMC Bioinformatics. 12, 323 (2011).</li><li>White, B. N., De Lucca, F. L., Turner, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+analysis+of+RNA.">Preparation and analysis of RNA.</a> Analytical Biochemistry of Insects. , (1977).</li><li>Hawley, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+Aedes+albopictus.">The biology of Aedes albopictus.</a> J. Am. Mosq. Contr. Assoc. 4, 1-39 (1988).</li><li>Dowling, Z., Ladeau, S. L., Armbruster, P., Biehler, D., Leisnham, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Socioeconomic+status+affect+mosquito+(Diptera:Culicidae)+larval+habitat+type+availability+and+infestation+level.">Socioeconomic status affect mosquito (Diptera:Culicidae) larval habitat type availability and infestation level.</a> J. Med Entomol. 50, 764-772 (2013).</li><li>Benedict, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2,4,10.+Bloodfeeding:+Membrane+apparatuses+and+animals.">Chapter 2,4,10. Bloodfeeding: Membrane apparatuses and animals.</a> Methods in Anopheles Research. , (2010).</li><li>Das, S., Garver, S., Ramirez, J. R., Xi, Z., Dimopolous, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+dengue+infections+in+mosquitoes+(A.+aegypti)+and+infection+phenotype+determination.">Protocol for dengue infections in mosquitoes (A. aegypti) and infection phenotype determination.</a> J. Vis. Exp. 5 (220), (2007).</li><li>Goff, S. 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=The+iPlant+collaborative:+cyberinfrastructure+for+plant+biology.">The iPlant collaborative: cyberinfrastructure for plant biology.</a> Plant Sci. 2 (34), (2011).</li><li>Robinson, M. D., McCarthy, D. J., Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=edgeR:+a+Bioconductor+package+for+differential+expression+analysis+of+digital+gene+expression+data.">edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.</a> Bioinformatics. 26, 139-140 (2010).</li><li>Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UCHIME+improves+sensitivity+and+speed+of+chimera+detection.">UCHIME improves sensitivity and speed of chimera detection.</a> Bioinformatics. 27 (16), 2194-2220 (2011).</li><li>Goecks, J., 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=Galaxy:+a+comprehensive+approach+for+supporting+accessible,+reproducible,+and+transparent+computational+research+in+the+life+sciences.">Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences.</a> Genome Biol. 11 (8), 86 (2010).</li></ol>

The authors have nothing to disclose.

该协议提出的方法来发现，由于差异表达基因在光周期诱导A.滞白纹伊蚊 。该协议是在于其独特地结合蚊子饲养和生物信息学技术，使访问新手分子生理学方案的所有实验方面显著 - 特别是对那些集中在光周期滞育反应。现有的方法，据我们所知，不提供尽可能多的细节，在饲养协议 - 这往往是要找准饲养的错误 - 他们也不提供在饲养阶段，这将使得下游的成功生物信息学分析的实?...

Rapid advances in next-generation sequencing (NGS) technologies are providing exciting opportunities to probe the molecular underpinnings of a wide range of genetically complex ecological adaptations in a broad diversity of non-model organisms1&#8211;3. This approach is extremely powerful because it establishes a basis for population and functional genomics studies of organisms with an especially interesting and/or well-described ecology or evolutionary history, as well as organisms of practical concern, such as agricultural pests and disease vectors. Thus, NGS technologies are leading to rapid advances in the fields of ecology and have the potential to address problems such as understanding the mechanistic bases of biological responses to rapid contemporary climate change4, the spread of invasive species5, and host-pathogen interactions6,7.
The extraordinary potential of NGS technologies for addressing basic and applied questions in ecology and evolutionary biology is in part due to the fact that these approaches can be applied to any organism at a moderate cost that is feasible for most research laboratories. Furthermore, these approaches provide genome-wide information without the requirement of a priori genetic resources such as a microarray chip or complete genome sequence. Nevertheless, to maximize the productivity of NGS experiments requires careful consideration of experimental design including issues such as the developmental timing and tissue-specificity of RNA sampling. Furthermore, the technical skills required to analyze the massive amounts of data produced by these experiments, often up to several hundred million DNA sequence reads, has been a particular challenge and has limited the widespread implementation of NGS approaches.
Recent RNA-Seq studies on the transcriptional bases of diapause in the invasive and medically important mosquito Aedes albopictus provide a useful example of some of the experimental protocols that can be employed to successfully apply NGS technology to studying the molecular basis of a complex ecological adaptation in a non-model organism8&#8211;10. A. albopictus is a highly invasive species that is native to Asia but has recently invaded North America, South America, Europe, and Africa11,12. Like many temperate insects, temperate populations of A. albopictus survive through winter by entering a type of dormancy referred to as photoperiodic diapause. In A. albopictus, exposure of pupal and adult females to short (autumnal) day lengths leads to the production of diapause eggs in which embryological development is completed, but the pharate larva inside the chorion of the egg enters a developmental arrest that renders the egg refractory to hatching stimulus15&#8211;17. Diapause eggs are more desiccation resistant5,18 and contain more total lipids19 than non-diapause eggs. Photoperiodic diapause in A. albopictus is thus a maternally controlled, adaptive phenotypic plasticity that is essential for surviving the harsh conditions of winter in temperate environments. Despite the well-understood ecological significance of photoperiodic diapause in a wide range of insects20,21, the molecular basis of this crucial adaptation is not well characterized in any insect22. In organisms such as A. albopictus that undergo an embryonic diapause at the pharate larval stage, it remains a particularly compelling challenge to understand how the photoperiodic signal received by the mother is passed to the offspring and persists through the course of embryonic development to cause arrest at the pharate larval stage.
This protocol describes mosquito rearing, experimental design and bioinformatics analyses for NGS experiments (transcriptome sequencing) performed to elucidate transcriptional components of photoperiodic diapause in A. albopictus. This protocol can be used for additional studies of diapause in A. albopictus, can be adapted to investigate diapause in other closely related species such as other aedine mosquitoes that undergo egg diapause23, and is also more generally relevant to employing NGS approaches to study the transcriptional bases of any complex adaptation in any insect.

<table><tbody><tr><td>Incubator - Model 818</td><td>Thermo-Scientific</td><td>3751</td><td>120 V</td></tr><tr><td>Controlled environment room</td><td>Thermax Scientific</td><td>N/A</td><td>Walk-in controlled environment room built to custom specifications by Thermax Scientific Products. A larger alternative to an incubator. http://thermmax.com/</td></tr><tr><td>Cool Fluorescent bulb</td><td>Philips</td><td>392183</td><td>4 W</td></tr><tr><td>Petri Dish 100 mm x 20 mm</td><td>Fisher</td><td>08-772-E</td><td></td></tr><tr><td>Filter Paper 20.5 cm</td><td>Fisher</td><td>09-803-6J</td><td></td></tr><tr><td>9.5 L Bucket</td><td>Plastican</td><td>Bway Products</td><td>http://www.bwayproducts.com/sites/portal/plastic-products/plastic-open-head-pails/117</td></tr><tr><td>Utility Fabric-Mosquito Netting White</td><td>Joann</td><td>10173292</td><td>http://www.joann.com/utility-fabric-mosquito-netting-white/10173292.html</td></tr><tr><td>Orthopedic stockings</td><td>Albahealth</td><td>23650-040</td><td>product no. 081420</td></tr><tr><td>Organic Raisins</td><td>Newman&#039;s Own</td><td>UPC: 884284040255</td><td></td></tr><tr><td>Oviposition cups (brown)</td><td>Fisher Scientific</td><td>03-007-52</td><td>The product is actually an amber 125 ml bottle that we saw the top off of.</td></tr><tr><td>Recycled Paper Towels</td><td>Seventh Generation</td><td>30BPT120</td><td></td></tr><tr><td>Modular Mates Square Tupperware Set</td><td>Tupperware</td><td></td><td>http://order.tupperware.com/pls/htprod_www/coe$www.add_items</td></tr><tr><td>Glass Grinder</td><td>Corning Incorporated</td><td>7727-2</td><td>These Tenbroeck tissue grinders break the eggs and release RNA into the TRI Reagent.</td></tr><tr><td>TRI Reagent</td><td>Sigma Aldrich</td><td>T9424</td><td>Apply 1 ml TRI Reagent per 50-100 mg of tissue. Caution &amp;#8212; this reagent is toxic.</td></tr><tr><td>TURBO DNA-free</td><td>Ambion/Life Technologies</td><td>AM1907</td><td>This kit generates greater yield than traditional DNase treatment followed by phenol/chloroform cleanup, and it is simpler to use.</td></tr><tr><td>RNaseZap</td><td>Ambion/Life Technologies</td><td>AM9782</td><td>Apply liberally on the bench surfaces and any equipment that might be in contact with the RNA samples. The solution is slightly alkaline/corrosive, can cause irritation and is harmful when swallowed.</td></tr><tr><td>2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2939AA</td><td>Place up to 12 RNA samples on one chip.</td></tr><tr><td>Hemotek Membrane Feeder</td><td>Hemotek&amp;nbsp;</td><td>5W1</td><td>This system&amp;nbsp; provides 5 feeding stations that can be used simultaneously. Includes PS5 Power Unit and Power cord; 5 FUI Feeders + Meal Reservoirs and O-rings; Plastic Plugs, Hemotek collagen feeding membrane; Temperature setting tool; and Plug extracting tool. The company&#039;s mailing address is: Hemotek Ltd; Unit 5 Union Court; Alan Ramsbottom Way; Great Harwood; Lancashire, UK; BB6 7FD; tel: +44 1254 889 307.</td></tr><tr><td>Digital Thermometer and Probe</td><td>Hemotek&amp;nbsp;</td><td>MT3KFU</td><td>MicroT3 thermometer and KFU probe. This is used to set the temperature of each FUI feeding unit.</td></tr><tr><td>Chicken Whole Blood, non-sterile with Sodium Citrate</td><td>Pel-Freez Biologicals</td><td>33130-1</td><td>The 500 ml of blood were frozen and stored in 20 ml aliquots at -80 &amp;#176;C for up to 1 year. Thaw blood at room temperature for at least 1 hr before using.</td></tr></tbody></table>

an experimental and bioinformatics protocol for rna-seq analyses of photoperiodic diapause in the asian tiger mosquito, aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

两个有代表性的RNA样品荧光显示两个频段约2000新台币（ 图1A，B）。昆虫28S核糖体RNA是由通过氢键，这很容易被简单加热或扰乱氢键35代理人打乱在一起举行了两次多核苷酸链。将所得的两组件是大致相同的尺寸的18S核糖体RNA。第二RNA样品显示出高程度的退化（ 图1B）。 光周期处理一组有代表性A.的伊蚊蚊子导致高滞育发病饲养?...

Watch this Scientific Journal Video about An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus at JoVE.com

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，<em&gt;白纹伊蚊</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

an-experimental-bioinformatics-protocol-for-rna-seq-analyses

Research

JoVE Journal

Biology

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

13.2K 次观看.  Georgetown University. RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data. 

视频: An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

成蚊（冈比亚按蚊的RNAi实验议定书）

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

科研

生物学

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

可重复的 dsrna 微注射和蚊子和家蝇的奥维恩生物测定

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

环境科学

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and homing endonucleases (HEs). More recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has offered an easier and cheaper alternative for precision genome engineering. Following nuclease action, DNA repair pathways will fix the broken DNA ends, often introducing indels. These out-of-frame mutations are then used for understanding gene function in the target organisms. A drawback, however, is that mutant individuals carry no dominant marker, making identification and tracking of mutant alleles challenging, especially at scales needed for many experiments. 
High-resolution melt analysis (HRMA) is a simple method to identify variations in nucleic acid sequences and utilizes PCR melting curves to detect such variations. This post-PCR analysis method uses fluorescent double-stranded DNA-binding dyes with instrumentation that has temperature ramp control data capture capability and is easily scaled to 96-well plate formats. Described here is a simple workflow using HRMA for the rapid detection of CRISPR/Cas9-induced indels and the establishment of mutant lines in the mosquito Ae. aegypti. Critically, all steps can be performed with a small amount of leg tissue and do not require sacrificing the organism, allowing genetic crosses or phenotyping assays to be performed after genotyping.

Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti

This article details a protocol for rapid identification of indels induced by CRISPR/Cas9 and selection of mutant lines in the mosquito Aedes aegypti using high-resolution melt analysis.

使用埃及伊蚊的高分辨率熔融分析进行CRISPR/Cas9诱变后的Indel检测

Mosquito gene editing has become routine in several laboratories with the establishment of systems such as transcription-activator-like effector nucleases ...

遗传学

Electroporation-mediated RNA Interference Method in Odonata

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, morphology, and behavior drastically from aquatic larvae to terrestrial/aerial adults without pupal stage. Odonata adults have a well-developed color vision and show a remarkable diversity in body colors and patterns across sexes, stages, and species. While many ecological and behavioral studies on Odonata have been conducted, molecular genetic studies have been scarce mainly due to the difficulty in applying gene functional analysis to Odonata. For instance, RNA interference (RNAi) is less effective in the Odonata, as reported in the Lepidoptera. To overcome this problem, we successfully established an RNAi method combined with in vivo electroporation. Here we provide a detailed protocol including a video of the electroporation-mediated RNAi method as follows: preparation of larvae, species identification, preparation of dsRNA/siRNA solution and injection needles, ice-cold anesthesia of larvae, dsRNA/siRNA injection, in vivo electroporation, and individual rearing until adult emergence. The electroporation-mediated RNAi method is applicable to both damselflies (suborder Zygoptera) and dragonflies (suborder Anisoptera). In this protocol, we present the methods for the blue-tailed damselfly Ischnura senegalensis (Coenagrionidae) as an example of damselfly species and the pied skimmer dragonfly Pseudothemis zonata (Libellulidae) as another example of dragonfly species. As representative examples, we show the results of RNAi targeting the melanin synthesis gene multicopper oxidase 2. This RNAi method will facilitate understanding of various gene functions involved in metamorphosis, morphogenesis, color pattern formation, and other biological features of Odonata. Moreover, this protocol may be generally applicable to non-model organisms in which RNAi is less effective in gene suppression due to the inefficiency and low penetrance.

We provide a detailed protocol for electroporation-mediated RNA interference in insects of the order Odonata (dragonflies and damselflies) using the blue-tailed damselfly (Ischnura senegalensis: Coenagironidae: Zygoptera) and the pied skimmer dragonfly (Pseudothemis zonata: Libellulidae: Anisoptera).

奥多纳塔电介质介质RNA干扰法

Dragonflies and damselflies (order Odonata) represent one of the most ancestral insects with metamorphosis, in which they change their habitat, ...

发育生物学

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi介导的基因敲除和<em&gt;在体内</em在成年女性利尿含量<em&gt;埃及伊蚊</em&gt;蚊子

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

生理录音和黄热病蚊子的味觉附属物RNA测序<em&gt;埃及伊蚊</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAi的触发传送到<em&gt;冈比亚按蚊</em&gt;蛹

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

IR-TEx: An Open Source Data Integration Tool for Big Data Transcriptomics Designed for the Malaria Vector Anopheles gambiae

IR-TEx is an application written in Shiny (an R package) that allows exploration of the expression of (as well as assigning functions to) transcripts whose expression is associated with insecticide resistance phenotypes in Anopheles gambiae mosquitoes. The application can be used online or downloaded and used locally by anyone. The local application can be modified to add new insecticide resistance datasets generated from multiple -omics platforms. This guide demonstrates how to add new datasets and handle missing data. Furthermore, IR-TEx can be completely and easily recoded to use-omics datasets from any experimental data, making it a valuable resource to many researchers. The protocol illustrates the utility of IR-TEx in identifying new insecticide resistance candidates using the the microsomal glutathione transferase, GSTMS1, as an example. This transcript is upregulated in multiple pyrethroid resistant populations from C&#244;te D&#39;Ivoire and Burkina Faso. The identification of co-correlated transcripts provides further insight into the putative roles of this gene.

IR-TEx: An Open Source Data Integration Tool for Big Data Transcriptomics Designed for the Malaria Vector Anopheles gambiae

IR-TEx explores insecticide resistance-related transcriptional profiles in the species Anopheles gambiae. Provided here are full instructions for using the application, modifications for exploring multiple transcriptomic datasets, and using the framework to build an interactive database for collections of transcriptomic data from any organism, generated in any platform.

IR-TEx:专为疟疾病媒阿诺菲斯甘巴病设计的大数据转录组学的开源数据集成工具

IR-TEx is an application written in Shiny (an R package) that allows exploration of the expression of (as well as assigning functions to) transcripts ...

A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus on one or more gene families that are significant to the organism and study. In this protocol, we outline bioinformatic steps to generate a phylogeny and to quantify the expression of genes of interest. Phylogenetic trees can give insight into how genes are evolving within and between species as well as reveal orthology. These results can be enhanced using RNA-seq data to compare the expression of these genes in different individuals or tissues. Studies of molecular evolution and expression can reveal modes of evolution and conservation of gene function between species. The characterization of a gene family can serve as a springboard for future studies and can highlight an important gene family in a new genome or transcriptome paper.

The purpose of this protocol is to investigate the evolution and expression of candidate genes using RNA sequencing data.

使用RNA-seq研究分子进化和基因表达的生物信息学管道

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

用于下一代测序的基于磁珠的蚊子DNA提取协议

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，

摘要

探索更多视频

此视频中的章节