Validation of reference genes for gene expression studies in the honey bee, Apis mellifera, by quantitative real-time RT-PCRAnete Pedro Lourenço1, Aline Mackert1, Alexandre dos Santos Cristino2 and Zilá Luz Paulino Simões3
1 Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14040-900, Ribeirão Preto, SP, Brazil
2 Instituto de Matemática e Estatística, Universidade de São Paulo, Rua do Matão, 1010, 05508-090, Cidade Universitária, São Paulo, SP, Brazil
3 Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14040-901, Ribeirão Preto, SP, Brazil
Received 8 October 2007 - Revised 28 January 2008 - Accepted 14 February 2008 - Published online 28 May 2008
Abstract - For obtaining accurate and reliable gene expression results it is essential that quantitative real-time RT-PCR (qRT-PCR) data are normalized with appropriate reference genes. The current exponential increase in postgenomic studies on the honey bee, Apis mellifera, makes the standardization of qRT-PCR results an important task for ongoing community efforts. For this aim we selected four candidate reference genes (actin, ribosomal protein 49, elongation factor 1-alpha, tbp-association factor) and used three software-based approaches (geNorm, BestKeeper and NormFinder) to evaluate the suitability of these genes as endogenous controls. Their expression was examined during honey bee development, in different tissues, and after juvenile hormone exposure. Furthermore, the importance of choosing an appropriate reference gene was investigated for two developmentally regulated target genes. The results led us to consider all four candidate genes as suitable genes for normalization in A. mellifera. However, each condition evaluated in this study revealed a specific set of genes as the most appropriated ones.
Key words: quantitative real-time RT-PCR / reference genes / Apis mellifera / gene expression normalization
© INRA, DIB-AGIB, EDP Sciences 2008