Evaluating potential reference genes in a cultured cell line in Diabrotica undecimpunctata

Insect cell lines have been valuable model systems for enhancing our understanding of basic physiological processes in arthropods. However, basic information about cell lines under different experimental conditions is necessary to realize their full experimental potential, particularly in instances where relative quantitative PCR is used to assess changes in transcript levels. In this investigation, we assessed the stability of expression of six putative reference genes, including actin, tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha, and 40 S ribosomal protein (RpS9) in cultured cells of Diabrotica undecumpunctata (SCR), a significant crop pest. RNA was harvested from SCR cells under different treatment conditions, including treatment with double-stranded RNA, and stability of reference gene transcript levels were assessed using the analysis programs RefFinder, NormFinder, gNorm, and BestKeeper. Our findings show that the best reference genes differed depending on the settings under which they were tested. Our results will provide a firm foundation for future use of SCR cells as a model system for enhancing our understanding of cellular physiology of insects.