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

If the addition of a gene produced large differences between consecutives V n/n+1 , then the added gene should be preferably included for calculation of NF n [3] . Following this criteria, only the IPNV infection assay on RTS11 cell cultures might consider the inclusion of a UBQ and EF1A were the best ranked reference gene candidates, as they had the lowest sum p values (0.813 and 1.023, respectively) which represents the standard deviation (SD) of reference gene expression over all infections investigated (Table 2 ). EF1A has been demonstrated to be a good housekeeping gene for the analysis of infection challenges, although a slight upregulation in RTS11 cells after 9 days P. salmonis infection ( Figure 5A ). In contrast to the reports of modulated expression by viral infections of human cell lines [26] and in infection of Atlantic salmon with ISAV [11] , ACTB showed in general a good performance during P. salmonis and IPNV infection, being considered in all cases as one of the three most stable genes ( Figure 4a ). As well, its reliability has been confirmed in LPS stimulated cells and tissues of fish [12] and birds [27] .