Reference genes evaluated for use in infectious pancreatic necrosis virus real-time RT-qPCR assay applied during different stages of an infection.

The stability of 6 reference genes, 18S, beta-actin, RPS20, eEF1alpha, G6PDH and GAPDH, was examined in tissues from Atlantic salmon (Salmo salar) and Chinook salmon embryo cells (CHSE-214). The main objective of this study was to determine the most suitable reference genes for use for the normalisation of data in quantitative real-time RT-qPCR assays conducted on infected tissues. The tissue samples selected for analysis were taken from head kidney and pylorus and collected at different time points during a challenge experiment with infectious pancreatic necrosis virus (IPNV). The stability of some of the reference genes was also studied in infected CHSE-214 cells. The ranking of the genes examined was carried out using the geNorm program. This program determines the most stable genes from a set of genes tested in a given cDNA sample. The stability of the reference genes varied in different tissues and in the cell line at different stages of infection with IPNV. This study demonstrated that tissue-specific combinations of reference genes must be used to normalise real time data for use for the quantitation of IPNV.