Evaluation of reference genes for insect olfaction studies

Abstract

BackgroundQuantitative 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.MethodsWe 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.ResultsWe 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0862-x) contains supplementary material, which is available to authorized users.

Molecular basis of peripheral olfactory plasticity in Rhodnius prolixus, a Chagas disease vector

“…A set of candidate reference genes (Table 1) was selected because they were all previously used for qPCR normalization in triatomines (Majerowicz et al, 2011;Paim et al, 2012) and other insect species (Scharlaken et al, 2008;Lord et al, 2010;Ling and Salvaterra, 2011;Ponton et al, 2011). Table 2 lists all reference factors calculated as the geometric means of the most stable combinations of these genes (Omondi et al, 2015b), used to evaluate changes in gene expression in the antennae of R. prolixus. The sequences of reference and target genes (RproOrco, RproIR8a, RproIR25a, and RproIR76b) were identified in the R. prolixus genome (available on www.vectorbase.org/organisms/rhodnius-prolixus) using a local tBLASTn algorithm (Altschul et al, 1997).…”

Deorphanization of an odorant receptor revealed new bioactive components for green mirid bug Apolygus lucorum (Hemiptera: Miridae)

“…In agreement with our findings, previous studies have also demonstrated that 1, 8‐Cineole is highly repellent against a diverse range of agricultural and medical pests . S ‐(−)‐ cis ‐Verbenol is a ubiquitously terpenoid volatile compound present in many plants and proved to be repellent for different insect pests . Behavioral responses of first‐instar nymphs of Chagas disease vectors, Rhodnius prolixus and Triatoma infestans (Heteroptera: Reduviidae) to several monoterpenes including S ‐(−)‐ cis ‐Verbenol were tested.…”

“…[55][56][57][58] S-(−)-cis-Verbenol is a ubiquitously terpenoid volatile compound present in many plants and proved to be repellent for different insect pests. [59][60][61][62] Behavioral responses of first-instar nymphs of Chagas disease vectors, Rhodnius prolixus and Triatoma infestans (Heteroptera: Reduviidae) to several monoterpenes including S-(−)-cis-Verbenol were tested. All tested chemicals were repellent for T. infestans and their efficiencies were as equal as DEET.…”

Genómica, proteómica y fisiología de neuropéptidos en el insecto triatomino Rhodnius prolixus

“…Todas las muestras se procesaron siguiendo las especificaciones detalladas en anexo para extracción de ARN y síntesis de ADNc. Se utilizaron como genes de referencia β-tubulina (β-tub) y factor de elongación-1 (RpEf1), que demostraron ser estables en R. prolixus bajo distintas condiciones (Majerowicz, Alves-Bezerra et al 2011;Omondi, Latorre-Estivalis et al 2015). La eficiencia de todos los primers utilizados en qRT-PCR fue evaluada a las concentraciones de molde: 1:2; 1:10 y 1:20 y un modelo de regresión lineal para determinarla.…”

“…Debido a esto se determinó utilizar ambos genes como housekeeping, realizando un promedio de los resultados de cada uno utilizando una media geométrica (Majerowicz, Alves-Bezerra et al 2011;Omondi, Latorre-Estivalis et al 2015).…”