Evaluation of Optimal Reference Genes for qRT-PCR Analysis in Hyphantria cunea (Drury)

SIMPLE SUMMARY: Reverse transcription-quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most accessible and widely-applied method for quantifying gene transcription. Stable reference genes need to be normalized to facilitate gene expression studies and obtain more accurate qRT-PCR data. In this research, eight reference genes from different functional categories and gene families were used to assess the stability level of gene expression under different conditions in H. cunea. Finally, the expression pattern of the target gene serine proteases (HcSP1) was evaluated under different conditions to verify the precision and reliability of the results. This study provides a comprehensive list of suitable reference genes for analyses and optional reference genes for other gene function research in H. cunea. ABSTRACT: The relative quantification of gene expression is mainly achieved through reverse transcription-quantitative PCR (qRT-PCR); however, its reliability and precision rely on proper data normalization using one or more optimal reference genes. Hyphantria cunea (Drury) has been an invasive pest of forest trees, ornamental plants, and fruit trees in China for many years. Currently, the molecular physiological role of reference genes in H. cunea is unclear, which hinders functional gene study. Therefore, eight common reference genes, RPS26, RPL13, UBI, AK, RPS15, EIF4A, β-actin, α-tub, were selected to evaluate levels of gene expression stability when subjected to varied experimental conditions, including developmental stage and gender, different tissues, larvae reared on different hosts and different larval density. The geNorm, BestKeeper, ΔCt method, and NormFinder statistical algorithms were used to normalize gene transcription data. Furthermore, the stability/suitability of these candidates was ranked overall by RefFinder. This study provides a comprehensive evaluation of reference genes in H. cunea and could help select reference genes for other Lepidoptera species.