An evaluation of potential reference genes for stability of expression in two salmonid cell lines after infection with either Piscirickettsia salmonis or IPNV

Peña, Andrea A; Bols, Niels C; Marshall, Sergio H

The present study aims to validate the usefulness of five potential housekeeping genes for normalization of a number of salmonid relevant immune genes. We are currently developing SYBR Green based real-time assays for studying the host immune response influenced by the infection with the facultative bacterium Piscirickettsia salmonis and with the IPNV, respectively. The in vitro models CHSE-214 (an epithelial-like embryo cell line derived from Chinook salmon, Oncorhynchus tshawitscha) and RTS11 (monocyte/macrophage-like cell line derived from rainbow trout, Oncorhynchus mykiss) have been of great help for this purpose because they have been shown to be susceptible to a wide range of viral infections [7, 8] and to P. salmonis [9, 10] . The potential reference genes we have examined are betaactin (ACTB), elongation factor 1-alpha (EF1A) and glyceraldehyd-3-phosphate dehydrogenase (GAPDH), which have been previously validated in several studies on diverse fish species including salmonids [11] [12] [13] , and ubiquitin (UBQ) and tubulin alpha (TUBA), which have been reported for fish species like the three-spine-stickelback (Gasterosteus aculeatus [14] ) and zebrafish (Danio rerio [15] ), but not in salmonids. The five housekeeping genes were selected based on their previous use as internal controls for gene expression studies, the availability of housekeeping gene sequences for salmonids and related teleost species, and because they have roles in different cellular functions (Table 1) , thus reducing the likelihood that they exhibited regulated covariation.