Evaluation of reference genes for insect olfaction studies

Particle and Fibre Toxicology

Marcelo Gustavo Lorenzo²  Rickard Ignell¹  Ivana Helena Rocha Oliveira²  Jose Manuel Latorre-Estivalis²  Bonaventure Aman Omondi³ 

[1] Chemical Ecology Unit, Department of Plant Protection Biology, SLU, Alnarp, Sweden;Vector Behavior and Pathogen Interaction Group, Centro de Pesquisas René Rachou, FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil;Current address: Bioversity International, Consultative Group for International Agricultural Research, Bujumbura, Burundi

关键词: Olfaction and triatomines;    Normalization process;    Reference genes;    RT-qPCR;   

Others  :  1181787
DOI  :  10.1186/s13071-015-0862-x

 received in 2015-01-12, accepted in 2015-04-15,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Omondi et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

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