title: Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.
abstract: 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.
authors: Evolutionary Ecology and Genetics group, Biodiversity Research Centre, Earth and Life Institute, Université catholique de Louvain, Croix du Sud 4, Louvain-la-Neuve, Belgium.
authors: Alok
authors: A
authors: Arun
authors: Evolutionary Ecology and Genetics group, Biodiversity Research Centre, Earth and Life Institute, Université catholique de Louvain, Croix du Sud 4, Louvain-la-Neuve, Belgium.
authors: Véronique
authors: V
authors: Baumlé
authors: Evolutionary Ecology and Genetics group, Biodiversity Research Centre, Earth and Life Institute, Université catholique de Louvain, Croix du Sud 4, Louvain-la-Neuve, Belgium.
authors: Gaël
authors: G
authors: Amelot
authors: Evolutionary Ecology and Genetics group, Biodiversity Research Centre, Earth and Life Institute, Université catholique de Louvain, Croix du Sud 4, Louvain-la-Neuve, Belgium.
authors: Caroline M
authors: CM
authors: Nieberding
mesh_terms: D000818:Animals; D002080:Butterflies / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling; D017344:Genes, Insect; D056726:Genetic Association Studies; D008297:Male; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012724:Sex Attractants / Q000096:biosynthesis; D012903:Smell / Q000235:genetics*; D012984:Software
chemical_list: D012724:Sex Attractants