Selection and evaluation of reference genes for quantitative real-time PCR analysis in lac insect (Kerria lacca)

The screening and identification of suitable reference genes are key steps in experiments involving the quantitative real-time polymerase chain reaction. However, the reference genes of Kerria lacca, a lac insect with considerable economic significance, have not been investigated. In this study, we used five statistical algorithms (Delta Ct, geNorm, NormFinder, BestKeeper, and RefFinder) to evaluate the stability of eight candidate reference genes (18S rRNA, 28S rRNA, beta-ACT, EF1-alpha, RPL10, RPL13, RPS11, and RPS15) in different developmental stages and tissues of K. lacca and under dsRNA treatment. Our results showed that the most stable reference genes were 28S rRNA and RPS11 for the developmental stages, RPL13 and RPS11 for different tissues, and beta-ACT and EF1-alpha for dsRNA treatments. Although the target genes FAS and 17 beta-HSD showed consistent expression profiles (alone or in combination) when normalized to the expression levels of the stable genes, those changed when the least stable reference gene (18S rRNA) was used. These results highlight that suitable reference genes should be chosen according to the specific experimental conditions used. The reliability and stability of different reference genes can provide a firm foundation for gene expression analysis in lac insects.