Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust.
Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust.
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DOI：
10.1186/1471-2199-12-7
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发表时间：
2011-02-16
影响因子：
--
通讯作者：
Sword GA
中科院分区：
文献类型：
--
作者：
Chapuis MP;Tohidi-Esfahani D;Dodgson T;Blondin L;Ponton F;Cullen D;Simpson SJ;Sword GA
澳大利亚鼠疫蝗虫，Chortoicumberterminifera，是最有前途的物种之一，解开套件的基因，从害羞和神秘的孤独行为的密度依赖性转变为高度活跃和聚集的群居行为，是蝗虫的特点。这是因为它缺乏其他蝗虫物种中伴随相变的颜色和形态方面的许多主要表型变化。逆转录-定量聚合酶链反应（RT-qPCR）是可用于确定基因表达变化的最灵敏方法。然而，为了准确地监测靶基因的表达，必须选择适当的标准化策略来控制样品之间的非特异性变异。在这里，我们确定了八个潜在的参考基因，并研究其表达的稳定性，在不同饲养密度处理的神经组织中的澳大利亚蝗灾。利用蝗虫中新的直链DNA序列，我们开发了编码18SrRNA、核糖体蛋白L32（RpL32）、犰狳（Arm）、肌动蛋白5C（Actin）、琥珀酸脱氢酶（SDHa）、甘油醛-3 β-脱氢酶（GAPDH）、延伸因子1 α（EF1a）和膜联蛋白IX（AnnIX）的基因的引物。这8个基因的相对转录水平，然后在不同的饲养密度（隔离，短期和长期拥挤），每个由5个池的4个神经组织样品从5龄幼虫的三个处理组进行了分析。SDHa和GAPDH都参与代谢途径，被鉴定为表达水平最不稳定，挑战了它们在正常化中的有用性。基于计算与geNorm和Normandy程序，最好的组合，两个基因的基因表达数据的标准化后，拥挤在澳大利亚鼠疫蝗虫是EF1a和臂。我们应用它们来研究一个靶基因，编码的Ca2+结合糖蛋白，Ca2+，这是以前发现的上调在大脑中的群居沙漠蝗虫，Schistocerca gregaria。有趣的是，该基因的表达并没有以同样的方式在两种蝗虫的大脑中随饲养密度而变化。与S. gregaria，在澳大利亚蝗灾中没有任何拥挤处理的效果。臂和EF1a是最稳定表达的两个参考基因的八个检查的RT-qPCR测定研究密度依赖性的行为变化在澳大利亚蝗灾可靠的标准化的组合。这样的标准化使我们能够表明，C。在沙漠蝗S.聚集。这种密度依赖的基因调控的比较结果提供了深入了解蝗虫的群居行为和大规模迁移的演变。我们评估的八个确定的基因也是用于涉及其他Oedipodinae物种的实验中使用的标准化基因的候选人，但基因稳定性的排名顺序必须根据具体情况确定。
The Australian plague locust, Chortoicetes terminifera, is among the most promising species to unravel the suites of genes underling the density-dependent shift from shy and cryptic solitarious behaviour to the highly active and aggregating gregarious behaviour that is characteristic of locusts. This is because it lacks many of the major phenotypic changes in colour and morphology that accompany phase change in other locust species. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive method available for determining changes in gene expression. However, to accurately monitor the expression of target genes, it is essential to select an appropriate normalization strategy to control for non-specific variation between samples. Here we identify eight potential reference genes and examine their expression stability at different rearing density treatments in neural tissue of the Australian plague locust. Taking advantage of the new orthologous DNA sequences available in locusts, we developed primers for genes encoding 18SrRNA, ribosomal protein L32 (RpL32), armadillo (Arm), actin 5C (Actin), succinate dehydrogenase (SDHa), glyceraldehyde-3P-dehydrogenase (GAPDH), elongation factor 1 alpha (EF1a) and annexin IX (AnnIX). The relative transcription levels of these eight genes were then analyzed in three treatment groups differing in rearing density (isolated, short- and long-term crowded), each made up of five pools of four neural tissue samples from 5th instar nymphs. SDHa and GAPDH, which are both involved in metabolic pathways, were identified as the least stable in expression levels, challenging their usefulness in normalization. Based on calculations performed with the geNorm and NormFinder programs, the best combination of two genes for normalization of gene expression data following crowding in the Australian plague locust was EF1a and Arm. We applied their use to studying a target gene that encodes a Ca2+ binding glycoprotein, SPARC, which was previously found to be up-regulated in brains of gregarious desert locusts, Schistocerca gregaria. Interestingly, expression of this gene did not vary with rearing density in the same way in brains of the two locust species. Unlike S. gregaria, there was no effect of any crowding treatment in the Australian plague locust. Arm and EF1a is the most stably expressed combination of two reference genes of the eight examined for reliable normalization of RT-qPCR assays studying density-dependent behavioural change in the Australian plague locust. Such normalization allowed us to show that C. terminifera crowding did not change the neuronal expression of the SPARC gene, a gregarious phase-specific gene identified in brains of the desert locust, S. gregaria. Such comparative results on density-dependent gene regulation provide insights into the evolution of gregarious behaviour and mass migration of locusts. The eight identified genes we evaluated are also candidates as normalization genes for use in experiments involving other Oedipodinae species, but the rank order of gene stability must necessarily be determined on a case-by-case basis.
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影响因子：
56.9
作者：
Furlong, EEM;Andersen, EC;Scott, MP
通讯作者：
Scott, MP
影响因子：
--
作者：
Infante, Carlos;Asensio, Esther;Manchado, Manuel
通讯作者：
Manchado, Manuel
影响因子：
2.7
作者：
Dheda, K;Huggett, JF;Zumla, A
通讯作者：
Zumla, A
影响因子：
2.2
作者：
Cullen, Darron A.;Sword, Gregory A.;Simpson, Stephen J.
通讯作者：
Simpson, Stephen J.
DOI：
10.1073/pnas.95.22.13052
发表时间：
1998-10-27
影响因子：
11.1
作者：
Collett, M;Despland, E;Krakauer, DC
通讯作者：
Krakauer, DC