Abstract
Honey bees are both important pollinators and model insects due to their highly developed sociality and colony management. To better understand the molecular mechanisms underlying honey bee colony management, it is important to investigate the expression of genes putatively involved in colony physiology. Although quantitative real-time PCR (qRT-PCR) can be used to quantify the relative expression of target genes, internal reference genes (which are stably expressed across different conditions) must first be identified to ensure accurate normalisation of target genes. To identify reliable reference genes in honey bee (Apis mellifera) colonies, therefore, we evaluated seven candidate genes (ACT, EIF, EF1, RPN2, RPS5, RPS18 and GAPDH) in samples collected from three honey bee tissue types (head, thorax and abdomen) across all four seasons using three analysis programmes (NormFinder, BestKeeper and geNorm). Subsequently, we validated various normalisation methods using each of the seven reference genes and a combination of multiple genes by calculating the expression of catalase (CAT). Although the genes ranked as the most stable gene were slightly different on conditions and analysis methods, our results suggest that RPS5, RPS18 and GAPDH represent optimal honey bee reference genes for target gene normalisation in qRT-PCR analysis of various honey bee tissue samples collected across seasons.
Highlights
The Western honey bee, Apis mellifera L., plays an important role as a p ollinator[1]
All PCR products amplified with each primer set showed a single band in 1% agarose gels and a shark single peak detected in the melting curve by RT-PCR
In order to find optimal reference genes for quantitative real-time PCR (qRT-PCR) assay in the honey bee across four seasons and three tissue types, we evaluated the expression stabilities of seven candidate reference genes, ACT,eukaryotic translation initiation factor (EIF), elongation factor 1 (EF1), RPN2, ribosomal protein S5 (RPS5), ribosomal protein S18 (RPS18) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using three analysis programmes: NormFinder, BestKeeper and geNorm
Summary
The Western honey bee, Apis mellifera L., plays an important role as a p ollinator[1]. Previous studies have demonstrated that endocrine system status and gene expression are important factors for flexible honey bee colony management, which involves colonies seasonally regulating their labour division and population dynamics[3,4,5]. QRT-PCR would be an ideal method for analysing the expression patterns of honey bee genes putatively involved in the plasticity of colony molecular physiology in samples collected across different seasons and tissues. Given the importance of accurate normalisation in qRT-PCR assays, reference genes have been identified and validated in various insect s pecies[8,13,14]. Seasonal expression stabilities of candidate reference genes have been compared between forager and nurse head in our previous study[7]. The seven reference genes and a combination of multiple references were validated by normalising catalase (CAT)expression
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