Validation of Housekeeping Genes for Gene Expression Analysis in Iwagaki Oyster (Crassostrea nippona) Under Salinity Stress by Quantitative Real-Time PCR

Hypo-salinity can reduce the immunological reaction in Crassostrea nippona, even lead to massive mortality. It is important to understand the molecular mechanism of oyster defense system, while quantitative real-time PCR can be employed in the study. However, the accuracy of quantitative real-time PCR relies on the use of suitable reference genes. In this study, the expression stability of 14 candidate reference genes including traditional housekeeping genes EF1A, TUB, TUA, GAPDH, RO21, as well as new candidate reference genes RPL5, RPL8, RPS27, RPL14, RPL4, CO3, RPS8, RPS4, CYTB in different tissues of C. nippona under salinity stress has been validated by quantitative real-time PCR. Ribosomal protein genes selected through expression analysis of transcriptome data from C. nippona generally were more stable than traditional reference genes. According to the geNorm analysis, RPL4 and RPS4 could be used as internal controls for studying gene expression in C. nippona with real-time PCR under salinity stress.