Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

Two different programs, geNorm and Normfinder, were used to analyze the data. According to geNorm, α-TUB + ACT5 are the most appropriate reference genes for gene expression profiling across the three different tissues in the female flies, while ACT3 + α-TUB are considered as the best for males. Furthermore, we evaluated the stability of the candidate reference genes to determine the sexual differences in the same tissue. In the midgut and Malpighian tubules, ACT2 + α-TUB are the best choice for both males and females. However, α-TUB + ACT1 are the best pair for fat body. Meanwhile, the results calculated by Normfinder are quite the same as the results with geNorm; α-TUB is always one of the most stable genes in each sample validated by the two programs.In this study, we validated the suitable reference genes for gene expression profiling in different tissues of B. dorsalis. Moreover, appropriate reference genes were selected out for gene expression profiling of the same tissues taking the sexual differences into consideration. This work not only formed a solid basis for future gene expression study in B. dorsalis, but also will serve as a resource to screen reference genes for gene expression studies in any other insects.Quantitative real-time reverse transcriptase PCR (RT-qPCR) has been widely used in gene expression analysis that provides insight into complex biological progresses [1]. This procedure of collecting data throughout the PCR process combines amplification and detection into a single step [2]. The advantages of this process include sensitivity, large dynamic range, and the potential for high throughout as well as accurate quantification [3].Although RT-qPCR is often described as the gold standard, there are still some limitations of this assay such as reverse transcription and normalization [4,5]. A common technique in RT-qPCR is to normalize data by measuring the expression of a reference gene in the same samples in parallel. Housekeeping genes such a