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In this study an important and often neglected aspect of gene expression studies in insects, the validation of appropriate reference genes with stable expression levels between sample groups, is addressed. Although in this paper the reference gene selection for the honeybee,  Apis mellifera  L. (Hymenoptera: Apidae) head was tested in the context of bacterial challenge with  Escherichia coli , this work can serve as a resource to help select and screen insect reference genes for gene expression studies in any tissue and under any experimental manipulation. Since it is recommended to use multiple reference genes for accurate normalization, we analyzed the expression of eleven candidate reference genes in the honeybee head, for their potential use in the analysis of differential gene expression following bacterial challenge. Three software programs, BestKeeper, Normfinder and geNorm, were used to assess candidate reference genes. GeNorm recommended the use of four reference genes. Both geNorm and Normfinder identified the genes  GAPDH, RPS18, actin  and  RPL13a  as the most stable ones, only differing in their ranking order. BestKeeper identified  RPS18  as being the reference gene with the least overall variation, but also  actin  and  GAPDH  were found to be the second and third most stable expressed gene. By a combination of three software programs the genes  actin, RPS18 and GAPDH  were found suitable reference genes in the honeybee head in the context of bacterial infection.

journal of insect science

Reference Gene Selection for Insect Expression Studies Using Quantitative Real-Time PCR: The Head of the Honeybee,<i>Apis mellifera</i>, After a Bacterial Challenge

Reference Gene Selection for Insect Expression Studies Using Quantitative Real-Time PCR: The Head of the Honeybee,Apis mellifera, After a Bacterial Challenge