title: Evaluation of reference genes for insect olfaction studies.
abstract: BACKGROUND
  Quantitative reverse transcription PCR (qRT-PCR) is a robust and accessible method to assay gene expression and to infer gene regulation. Being a chain of procedures, this technique is subject to systematic error due to biological and technical limitations mainly set by the starting material and downstream procedures. Thus, rigorous data normalization is critical to grant reliability and repeatability of gene expression quantification by qRT-PCR. A number of 'housekeeping genes', involved in basic cellular functions, have been commonly used as internal controls for this normalization process. However, these genes could themselves be regulated and must therefore be tested a priori.
  
  
  METHODS
  We evaluated eight potential reference genes for their stability as internal controls for RT-qPCR studies of olfactory gene expression in the antennae of Rhodnius prolixus, a Chagas disease vector. The set of genes included were: α-tubulin; β-actin; Glyceraldehyde-3-phosphate dehydrogenase; Eukaryotic initiation factor 1A; Glutathione-S-transferase; Serine protease; Succinate dehydrogenase; and Glucose-6-phosphate dehydrogenase. Five experimental conditions, including changes in age,developmental stage and feeding status were tested in both sexes.
  
  
  RESULTS
  We show that the evaluation of candidate reference genes is necessary for each combination of sex, tissue and physiological condition analyzed in order to avoid inconsistent results and conclusions. Although, Normfinder and geNorm software yielded different results between males and females, five genes (SDH, Tub, GAPDH, Act and G6PDH) appeared in the first positions in all rankings obtained. By using gene expression data of a single olfactory coreceptor gene as an example, we demonstrated the extent of changes expected using different internal standards.
  
  
  CONCLUSIONS
  This work underlines the need for a rigorous selection of internal standards to grant the reliability of normalization processes in qRT-PCR studies. Furthermore, we show that particular physiological or developmental conditions require independent evaluation of a diverse set of potential reference genes.
authors.affiliation: Chemical Ecology Unit, Department of Plant Protection Biology, SLU, Alnarp, Sweden. amanlgb@gmail.com.|Current address: Bioversity International, Consultative Group for International Agricultural Research, Bujumbura, Burundi. amanlgb@gmail.com.
authors.forename: Bonaventure Aman
authors.initials: BA
authors.lastname: Omondi
authors.affiliation: Vector Behavior and Pathogen Interaction Group, Centro de Pesquisas René Rachou, FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil. jose.estivalis@cpqrr.fiocruz.br.
authors.forename: Jose Manuel
authors.initials: JM
authors.lastname: Latorre-Estivalis
authors.affiliation: Vector Behavior and Pathogen Interaction Group, Centro de Pesquisas René Rachou, FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil. ivanoli@hotmail.com.
authors.forename: Ivana Helena
authors.initials: IH
authors.lastname: Rocha Oliveira
authors.affiliation: Chemical Ecology Unit, Department of Plant Protection Biology, SLU, Alnarp, Sweden. rickard.ignell@slu.se.
authors.forename: Rickard
authors.initials: R
authors.lastname: Ignell
authors.affiliation: Vector Behavior and Pathogen Interaction Group, Centro de Pesquisas René Rachou, FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil. marcelo@cpqrr.fiocruz.br.
authors.forename: Marcelo Gustavo
authors.initials: MG
authors.lastname: Lorenzo
mesh_terms: D000818:Animals; D057506:Arthropod Antennae / Q000502:physiology; D004772:Entomology / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D020043:Genes, Essential; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D012239:Rhodnius / Q000235:genetics* / Q000502:physiology; D012903:Smell / Q000235:genetics*