A complete listing of IEDB's class hierarchy and its properties is available at http://www.immuneepitope.org/ ontology/index.html  Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon BACKGROUND: Salmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking. RESULTS: The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription levels of genes encoding 18S rRNA, S20 ribosomal protein, β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), and two paralog genes encoding elongation factor 1A (EF1A(A )and EF1A(B)) were quantified in gills, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine in six untreated adult fish, in addition to a group of individuals that went through smoltification. Based on calculations performed with the geNorm VBA applet, which determines the most stable genes from a set of tested genes in a given cDNA sample, the ranking of the examined genes in adult Atlantic salmon was EF1A(B)>EF1A(A)>β-actin>18S rRNA>S20>GAPDH. When the same calculations were done on a total of 24 individuals from four stages in the smoltification process (presmolt, smolt, smoltified seawater and desmoltified freshwater), the gene ranking was EF1A(B)>EF1A(A)>S20>β-actin>18S rRNA>GAPDH. CONCLUSION: Overall, this work suggests that the EF1A(A )and EF1A(B )genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon. In real-time RT-PCR, the expression levels of the target genes of interest are estimated on the basis of endogenous controls. Various housekeeping genes, ribosomal RNA (rRNA) and total RNA are most commonly used as references in gene expression analysis today. The purpose of these controls is to remove or reduce differences due to sampling, i.e. differences in RNA quantity and quality.