Reference Genes for Expression Analyses by QRT-PCR in (Lepidoptera: Gelechiidae)

Due to a lack of effective internal references, studies on functional genes in , a serious Lepidopteran pest attacking potatoes worldwide, have been greatly limited. To select suitable endogenous controls, ten housekeeping genes of actin (), α-tubulin (), glyceraldehyde-3-phosphate dehydrogenase (), elongation factor 1α (), and ribosomal RNA (, ), ribosomal protein genes , and and superoxide dismutase () were tested. Their expression levels were determined under three different experimental conditions (developmental stages, tissues/organs and temperatures) using qRT-PCR technology. The stability was evaluated with five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). The results clarified that , and are ranked as the best reference gene combination for measuring gene expression levels among different developing stages and under various temperatures; and are recommended to normalize the gene expression levels among diverse tissues. and are the best reference genes in all the experimental conditions. To validate the utility of the selected reference pair, and , we estimated the tissue-biased expression level of chitin synthase A gene (). As expected, was abundantly expressed in ectodermally derived epidermal cells, and lowly transcribed in the midgut. These findings will lay the foundation for future research on the molecular physiology and biochemistry of .