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Background  Due to the limited number of species specific antibodies against fish proteins, differential gene expression analyses are vital for the study of host immune responses. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most powerful tools for this purpose. Nevertheless, the accuracy of the method will depend on the careful selection of genes whose expression are stable and can be used as internal controls for a particular experimental setting.    Findings  The expression stability of five commonly used housekeeping genes [beta-actin ( ACTB ), elongation factor 1-alpha ( EF1A ), ubiquitin ( UBQ ), glyceraldehyd-3-phosphate dehydrogenase ( GAPDH ) and tubulin alpha ( TUBA )] were monitored in salmonid cell lines CHSE-214 and RTS11 after infection with two of the most fastidious fish pathogens, the facultative bacterium  Piscirickettsia salmonis  and the aquabirnavirus IPNV (Infectious Pancreatic Necrosis Virus). After geNorm analysis,  UBQ  and  EF1A  appeared as the most stable, although  EF1A  was slightly upregulated at late stages of  P. salmonis  infection in RTS11.  ACTB  instead, showed a good performance in each case, being always considered within the three most stable genes of the panel. In contrast, infection-dependent differential regulation of  GAPDH  and  TUBA  was also demonstrated.    Conclusion  Based on the data presented here with the cell culture models CHSE-214 and RTS11, we suggest the initial choice of  UBQ ,  ACTB  and  EF1A  as reference genes in qRT-PCR assays for studying the effect of  P. salmonis  and IPNV on the host immune response.

bmc research notes

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