Evaluation of Reference Genes for Quantitative Real-Time PCR in Honey Bee, Apis mellifera, Under Various Miticide Exposure Conditions

Abstract

Various miticides are being applied to apiaries to prevent mites. However, abuse of miticides could seriously damage bee health. To understand the physiological response of honey bees caused by inappropriate exposure to miticides, it is necessary to identify the marker genes whose expression alters in honey bees following exposure to miticides. Although quantitative real-time polymerase chain reaction (qRT-PCR) is widely used for gene expression analysis, selecting appropriate stably expressed reference gene(s) across various conditions is essential for accurately determining target gene expression levels. Therefore, this study assessed the expression stabilities of 10 candidate reference genes (RPS5, RPS18, GAPDH, ARF1, RAB1a, PPI, PGK, SDH, TBP, and EF1) using C_q distribution and four algorithm programs (NormFinder, BestKeeper, geNorm, and RefFinder). Subsequently, we validated various normalization methods using each of the 10 reference genes and a combination of multiple genes by calculating the expression of the target gene (SOD2). Based on the various analysis methods used in this study, RPS5 is suggested as the most optimal reference gene for qRT-PCR analysis in honey bees under multiple conditions of miticide exposure.