An experimental and bioinformatics protocol for RNA-Seq analyses of photoperiodic diapause in the Asian tiger mosquito, Aedes albopictus.
An experimental and bioinformatics protocol for RNA-Seq analyses of photoperiodic diapause in the Asian tiger mosquito, Aedes albopictus.
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DOI：
10.3791/51961
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发表时间：
2014-11
期刊：
影响因子：
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通讯作者：
Monica F. Poelchau;Xin Huang;Allison C. Goff;Julie A. Reynolds;P. Armbruster
中科院分区：
文献类型：
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作者：
Monica F. Poelchau;Xin Huang;Allison C. Goff;Julie A. Reynolds;P. Armbruster
光周期滞育是一种重要的适应，使个体能够通过一系列生理变化，最明显的是发育停滞和新陈代谢减少，来逃避严酷的季节性环境。通过RNA-Seq分析全球基因表达谱可以为了解光周期滞育的转录机制提供重要的见解。亚洲虎蚊白纹伊蚊（Aedes albopictus）因其易于饲养、易诱导滞育和基因组资源丰富而成为研究滞育转录基础的优秀生物。本文提出了鉴定白纹伊蚊滞育诱导的转录差异的一般实验工作流程。综述了蚊虫的饲养技术、诱导滞育和非滞育发育的条件、种群滞育率的估算方法、RNA提取和完整性评估等。处理来自Illumina测序仪的RNA-Seq数据的工作流程在差异表达基因列表中达到高潮。具有代表性的结果表明，该方案可用于有效鉴定白纹伊蚊由于光周期差异在转录水平上的差异调控基因。通过适当的调整，这一工作流程可以很容易地适应于研究其他蚊子滞育或其他重要生活史特征的转录基础。
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.