Selection and validation of reference genes for RT-qPCR analysis in Sf9 cell line infected by Heliothis virescens ascovirus 3h (HvAV-3h)

Abstract The real-time quantitative reverse transcription PCR (qRT-PCR) is an efficient and pervasive technique for analysis gene expression. Despite the fact that this technique has been extensively used to explore the gene functions, the stability of the reference genes still requires being validated at different experimental conditions. This research aims to confirm that the expression stability of seven candidates reference genes under different concentrations sterile hemolymph containing virus (2, 4, 6, 10 8 -fold) infects the Spodoptera frugiperda cells line (Sf9). The appropriate reference genes as endogenous controls were determined through four computational algorithms (ΔCt, NormFinder, BestKeeper, and geNorm) and an integrated online software RefFinder. According to results of this study, TUB and ACT1 were proved to be the most stable reference genes in E2 fold and E4 fold respectively. PPI was the best reference gene in E6 and E8 fold, 28S and TUB were the most stable pair reference genes through all samples. These results can facilitate basic researches of the molecular mechanism of HvAV-3h and also prove to be helpful for the standard RT-PCR method.