Evaluation of reference genes for insect olfaction studies

Bonaventure Aman Omondi, Jose Manuel Latorre-Estivalis, Ivana Helena Rocha Oliveira, Rickard Ignell, Marcelo Gustavo Lorenzo

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. We evaluated eight reference genes for their stability as internal controls of olfactory gene expression in the antennae of Rhodnius prolixus, a Chagas disease vector. Five experimental conditions, including changes in age, developmental stage and feeding status were tested in both sexes. 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 co-receptor gene as an example, we demonstrated the extent of changes expected using different internal standards. This work underlines the need for a rigorous selection of internal standards to grant the reliability of normalization processes in qRT-PCR studies. Furthermore, it is shown that particular physiological or developmental conditions require independent evaluation of a diverse set of potential reference genes.