Selection and validation of reference genes for functional studies in the Calliphoridae family.
The genera Cochliomyia and Chrysomya contain both obligate and saprophagous flies, which allows the comparison of different feeding habits between closely related species. Among the different strategies for comparing these habits is the use of qPCR to investigate the expression levels of candidate genes involved in feeding behavior. To ensure an accurate measure of the levels of gene expression, it is necessary to normalize the amount of the target gene with the amount of a reference gene having a stable expression across the compared species. Since there is no universal gene that can be used as a reference in functional studies, candidate genes for qPCR data normalization were selected and validated in three Calliphoridae (Diptera) species, Cochliomyia hominivorax Coquerel, Cochliomyia macellaria Fabricius, and Chrysomya albiceps Wiedemann . The expression stability of six genes ( Actin, Gapdh, Rp49, Rps17, α -tubulin, and GstD1) was evaluated among species within the same life stage and between life stages within each species. The expression levels of Actin, Gapdh, and Rp49 were the most stable among the selected genes. These genes can be used as reliable reference genes for functional studies in Calliphoridae using similar experimental settings.
Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil Departamento de Genética, Evolução e Bioagentes, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
Gisele Antoniazzi
GA
Cardoso
Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil Departamento de Genética, Evolução e Bioagentes, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
Cleverson Carlos
CC
Matiolli
Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil Departamento de Genética, Evolução e Bioagentes, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
Ana Maria Lima
AM
de Azeredo-Espin
Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo (USP), São Paulo, SP, Brazil ttorres@ib.usp.br.
Tatiana Teixeira
TT
Torres
D000818:Animals; D004175:Diptera / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D019143:Evolution, Molecular; D005260:Female; D015870:Gene Expression; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D007814:Larva / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D019476:Insect Proteins; D012333:RNA, Messenger
BestKeeper; Chrysomya; Cochliomyia; NormFinder; geNorm; gene expression