Selection and Verification of Reference Genes for Gene Expression Studies in Different Cell Lines of Golden Pompano (Trachinotus ovatus)

Abstract

The golden pompano snout (GPS) and head kidney (GPHK) cell lines have been proven to be meaningful tools for the study on pathogenic infections in vitro. In this study, we aimed to select the most stable reference genes from seven housekeeping genes (Actin, B2M, GAPDH, RPL13, EF1A, 18S and UBCE) applied to two cell lines of golden pompano (GPS and GPHK) under both normal physiological conditions and stimulated conditions of the lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (Poly I:C) relying on quantitative real-time PCR (qRT-PCR). Additionally, the raw Ct value resulting from the qRT-PCR was analyzed by the geNorm, NormFinder and BestKeeper algorithm, and the results indicated that expression for all candidate genes exhibited some discrepancy under different experimental conditions or cell types. As for the non-stimulated group, 18S and RPL13 were the most appropriate reference genes in GPS and GPHK cells, respectively. Nevertheless, the most suitable reference genes in GPS and GPHK cells, under the stimulation of LPS, were RPL13 and 18S, respectively, whereas after being stimulated with Poly I:C, UBCE and EF1A were recommended as the optimal candidates for GPS and GPHK cells, respectively. To be sure of the reliability of the selected reference genes, immune-related genes (ISG15, BCL2, IRF1 and IRF7) were chosen as target genes to normalize. The study will provide a direction for various golden pompano cell lines to screen appropriate reference genes, and will set the stage for the application of these cell lines in relevant research areas.