It is an irreversible process and is tightly mediated by a set of core genes, such as p53, caspases and IAP antagonists 12, 13, 14. Many substances, such as azadirachtin, abamectin, camptothecin, spinosad and ultraviolet, could induce apoptosis in Sf9 cells 6, 7, 8, 9.Īpoptosis is a normal physiological process for elimination of unwanted cells and works as a homeostatic mechanism in the development of multicellular organisms, and as the defense mechanism in response to a wide variety of stimuli 10, 11. Recently, Sf9 cells have also been used for research on apoptosis. Besides, it is also considered as a stable and comparable standardized evaluation model for insecticide cytotoxicology 5. It is one of the common cell lines used for recombinant protein expression using the baculovirus-insect cell expression system due to its superiority of higher protein yields than that of mammalian cell lines and Xenopus laevis oocytes expression systems 3, 4. The insect cell line Sf9 derived from fall armyworm moth Spodoptera frugiperda was widely used for biotechnological applications in both academic laboratories and industry 1, 2. Our results not only facilitate a more accurate normalization for RT-qPCR data, but also provide the reliable assurance for further studies of apoptotic mechanisms under different stimulus in Sf9 cells. The best choices for cells under different apoptosis conditions were listed: EF2 and EF1α for cells treated with azadirachtin RPL13 and RP元 for cells treated with camptothecin EF1α and β-1-TUB for cells irradiated under ultraviolet and EF1α and EF2 for combinational analyses of samples. Based on comprehensively analysis, the expression stability of selected genes varied in cells with different apoptotic stimuli. Five algorithms, including NormFinder, BestKeeper, Delta Ct method, geNorm, and RefFinder, were used for stability ranking. In this study, eight genes were selected for analyses of their suitability as references for normalizing RT-PCR data in Sf9 cells treated with apoptotic agents. However, the selection of reference genes influences the accuracy of results profoundly. Due to its simplicity, high sensitivity and reliable specificity, RT-qPCR has been used widespread for analyzing expression levels of target genes. Previous study have demonstrated that apoptosis occurred in Spodoptera frugiperda cultured Sf9 cells, which triggered by diverse apoptotic stimuli, including azadirachtin, camptothecin and ultraviolet. As a tightly controlled cell death process, apoptosis eliminates unwanted cells and plays a vital role in multicellular organisms.
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