Arun Alok, Baumlé Véronique, Amelot Gaël, Nieberding Caroline M
Evolutionary Ecology and Genetics group, Biodiversity Research Centre, Earth and Life Institute, Université catholique de Louvain, Croix du Sud 4, Louvain-la-Neuve, Belgium.
PLoS One. 2015 Mar 20;10(3):e0120401. doi: 10.1371/journal.pone.0120401. eCollection 2015.
Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression profile of the target candidate genes.
实时定量逆转录PCR（qRT-PCR）是一种广泛用于定量候选基因转录表达水平的技术。qRT-PCR需要选择一个或几个合适的内参基因，其表达谱在不同条件下保持稳定，以标准化候选基因的qRT-PCR表达谱。尽管几种蝴蝶物种（鳞翅目）已成为分子进化生态学中的重要模型，但迄今为止，尚无针对任何蝴蝶鉴定用于准确数据标准化的内参基因的研究。非洲灌木棕蝶（Bicyclus anynana）因其作为进化生态学模型的适用性而备受关注，我们在此首次进行了广泛研究，以鉴定该物种中合适的内参基因。我们监测了12个内参基因的表达谱：eEF-1α、FK506、UBQL40、RpS8、RpS18、HSP、GAPDH、VATPase、ACT3、TBP、eIF2和G6PD。我们测试了它们在三种不同组织（翅膀、大脑、触角）、两个发育阶段（蛹期和成虫期）以及两种性别（雄性和雌性）中的表达谱稳定性，所有这些样本都接受了两种食物处理（食物胁迫和自由采食对照）。使用基于两种算法的方法NormFinder和geNorm评估了12个内参基因的表达稳定性和排名。两种方法均确定RpS8是用于表达数据标准化的最合适内参基因。我们还表明，使用两个内参基因足以有效地标准化我们在非洲灌木棕蝶中使用的不同组织和实验条件下的qRT-PCR数据。最后，我们测试了选择稳定性不同的内参基因对非洲灌木棕蝶中参与嗅觉通讯的候选基因脂肪酸酰基还原酶2转录丰度标准化的影响，并且我们证实使用不稳定的内参基因会极大地改变目标候选基因的表达谱。
