Identification of suitable reference genes for quantitative gene expression analysis in clam Cyclina sinensis under salinity stress and Vibrio infection

The appropriate reference gene is a prerequisite for accurate normalization of gene expression level, and research on suitable reference genes in clam Cyclina sinensis is scarce. To improve the situation, we selected five commonly used housekeeping genes, including beta-actin, Elongation factor 1-alpha (EF1-alpha), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 40S ribosomal protein S18 (RPS18), and Tubulin alpha (TUB-alpha), then evaluated their expression stability in different adult tissues and under different experimental treatments (salinity stress and Vibrio parahaemolyticus infection). Their expression stability was analyzed by three frequently used programs, geNorm, NormFinder, and BestKeeper. This analysis indicated that multiple genes should be used for normalization, and we concluded that the reference gene combination GAPDH-RPS18-beta-actin, should be used for qRT-PCR analysis in different tissues of C. sinensis under normal physiological conditions. For the clams under salinity stress and Vibrio infection, EF1-alpha-GAPDH-RPS18 was recommended as the gene combination for qRT-PCR normalization. TUB-alpha was generally poorly ranked by all programs, and should not be used in future studies. This study should provide fundamental support for accurate quantitative gene expression analysis of this species.