Selection of Reference Genes for RT-qPCR Analysis in the Monarch Butterfly, Danaus plexippus (L.), a Migrating Bio-Indicator.
Selection of Reference Genes for RT-qPCR Analysis in the Monarch Butterfly, Danaus plexippus (L.), a Migrating Bio-Indicator.
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DOI：
10.1371/journal.pone.0129482
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Zhou X
中科院分区：
文献类型：
--
作者：
Pan H;Yang X;Bidne K;Hellmich RL;Siegfried BD;Zhou X
逆转录-定量聚合酶链式反应(RT-qPCR)是一种强有力的基因表达定量技术。为了便于基因表达研究并获得准确的结果，相对于稳定表达的参考基因的标准化是至关重要的。帝王蝶Danaus plexippus(L.)是最知名的昆虫物种之一，因为它每年在北美进行壮观的迁徙。除了它的伟大航程，丛枝螺旋藻还引起了人们对它作为生物指示器的作用的关注，从转基因生物(GMO)到自然生态系统。在本研究中，从竹节虫基因组中选取了9个参考基因作为候选参考基因。分别使用BestKeeper、Normfinder、GeNorm和ΔCt四个现成的计算程序评估了这些候选基因在不同生物和非生物条件下的表达谱。此外，RefFinder，一个整合了上述四种算法的网络计算平台，提供了这些参考基因稳定性的综合排名。结果，为每个实验条件推荐了一套参考基因。具体地说，在生物(发育、组织和性别)和非生物(光周期、温度和饲料α干扰)条件下，伸长因子1RF1RF1和核糖体蛋白49分别是最稳定的参考基因。随着最近2.73亿碱基对基因组草案的公布，这项研究的结果使我们能够建立标准化的RT-qPCR分析，并为随后的竹节虫基因组和功能基因组研究奠定基础。竹节虫是一种主要的生物指示物，也是一种新兴的迁徙动物模式。
Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique to quantify gene expression. To facilitate gene expression study and obtain accurate results, normalization relative to stably expressed reference genes is crucial. The monarch butterfly, Danaus plexippus (L.), is one of the most recognized insect species for its spectacular annual migration across North America. Besides its great voyages, D. plexippus has drawn attention to its role as a bio-indicator, ranging from genetically modified organisms (GMOs) to natural ecosystems. In this study, nine reference genes from D. plexippus genome were selected as the candidate reference genes. The expression profiles of these candidates under various biotic and abiotic conditions were evaluated using the four readily available computational programs, BestKeeper, Normfinder, geNorm, and ΔCt method, respectively. Moreover, RefFinder, a web-based computational platform integrating the four above mentioned algorisms, provided a comprehensive ranking of the stability of these reference genes. As a result, a suite of reference genes were recommended for each experimental condition. Specifically, elongation factor 1α (EF1A) and ribosomal protein 49 (RP49) were the most stable reference genes, respectively, under biotic (development, tissue, and sex) and abiotic (photoperiod, temperature, and dietary RNAi) conditions. With the recent release of a 273-million base pair draft genome, results from this study allow us to establish a standardized RT-qPCR analysis and lay a foundation for the subsequent genomic and functional genomic research in D. plexippus, a major bio-indicator and an emerging model for migratory animals.
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