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: a-tubulin; beta-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.

Keywords

RT-qPCR; Reference genes; Normalization process; Olfaction and triatomines

Published in

Parasites and Vectors
2015, Volume: 8, article number: 243

SLU Authors

• Bonaventure Omondi, Aman

  • Department of Plant Protection Biology, Swedish University of Agricultural Sciences
    

• Ignell, Rickard

  • Department of Plant Protection Biology, Swedish University of Agricultural Sciences
    

UKÄ Subject classification

Ecology
Biochemistry and Molecular Biology
Bioinformatics and Systems Biology


Publication identifier

DOI: https://doi.org/10.1186/s13071-015-0862-x

Permanent link to this page (URI)

https://res.slu.se/id/publ/69285