Reference gene selection for RT-qPCR analysis in Harmonia axyridis, a global invasive lady beetle

Harmonia axyridis is a voracious predator, a biological control agent, and one of the world most invasive insect species. The advent of next-generation sequencing platforms has propelled entomological research into the genomics and post-genomics era. Real-time quantitative PCR (RT-qPCR), a primary tool for gene expression analysis, is a core technique governs the genomic research. The selection of internal reference genes, however, can significantly impact the interpretation of RT-qPCR results. The overall goal of this study is to identify the reference genes in the highly invasive H. axyridis. Our central hypothesis is that the suitable reference genes for RT-qPCR analysis can be selected from housekeeping genes. To test this hypothesis, the stability of nine housekeeping genes, including 18S, 28S, ACTB, ATP1A1, GAPDH, HSP70, HSP90, RP49, and ATP6V1A, were investigated under both biotic (developmental time, tissue and sex), and abiotic (temperature, photoperiod, in vivo RNAi) conditions. Gene expression profiles were analyzed by geNorm, Normfinder, BestKeeper, and the ΔCt method. Our combined results recommend a specific set of reference genes for each experimental condition. With the recent influx of genomic information for H. axyridis, this study lays the foundation for an in-depth omics dissection of biological invasion in this emerging model.