Reference gene selection for qRT-PCR analysis of season- and tissue-specific gene expression profiles in the honey bee Apis mellifera

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. The Western honey bee, Apis mellifera L., plays an important role as a pollinator 1. In addition, the honey bee is considered to be a key model insect due to its relatively complex behaviours, including sociality, labour division and colony management 2. 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 3-5. In order to extend our understanding of the molecular mechanisms that underlie the regulation of honey bee colony physiology, information on the physiological functions of the genes putatively associated with colony management can be determined by analysing their expression profiles among different seasons and honey bee tissues 6,7. In quantitative real-time PCR (qRT-PCR), gene-specific mRNA (or cDNA) is quantified; this method has been used extensively because of its relative speed, sensitivity, replicability and accuracy 8,9. Therefore, 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. However, because qRT-PCR results are highly sensitive to the initial amount of RNA content in the amplification reaction, the interpretation of target gene expression levels among various conditions would result in appreciable errors without the use of a reliable internal standard 7-10. Therefore, prior to analysing target gene expression levels among conditions, reference genes are requir...

Glyphosate induces immune dysregulation in honey bees

“…The cycling conditions consisted of an initial cycle of 50 °C for 2 min and 95 °C for 2 min, followed by 40 cycles of a two-step PCR of 95 °C for 15 s and 60 °C for 1 min. Expression levels were measured in triplicate for each biological replicate and normalized against the housekeeping gene rps5 (Additional file 1 : Table S1) [ 71 ]. Relative expression was performed by means of the ΔΔC T method, and differences in expression between control and treatment groups were investigated using the linear regression ‘lm’ test in the pcr package [ 72 ] in R version 3.5.2 [ 70 ].…”

Pollen nutrition fosters honeybee tolerance to pesticides

“…All qPCR reactions were run in duplicate. The average cycle threshold values of vitellogenin were normalized to the geometric mean of the housekeeping genes actin and RPS18, which proved to have rather stable expression levels [70]. We used sequences of primers previously published [71,72].…”

Honey bee functional genomics using symbiont-mediated RNAi

“…qPCR primers for the gene of interest and reference gene are then tested for adequate efficiency. Subsequently, qPCR is performed on bee cDNA derived from the experimental group (gene of interest knocked down) and control groups (Positive control: inR1 or def1 knockdown; Negative control: gfp mock knockdown), using primers for the gene of interest and multiple reference genes (such as rps18 and gapdh ) 60,61 . Ct values determined by qPCR are used to calculate ΔΔCt, which is used to determine the relative expression of the gene of interest (compared to the reference gene) for the experimental and control groups 62,63 .…”

“…We use a target annealing temperature of 60 °C to match the qPCR primers previously designed for the reference gene rps18 18 . At this point, one should also order qPCR primers for reference bee genes such as rps18 and gapdh 60,61 . Primer sequences for using qPCR to monitor knockdown of the two example bee genes ( inR1 and def1 ), as well as for reference genes ( rps18 and gadph ), are shown below.…”