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

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.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.To date, cDNA microarray and quantitative real-time reverse transcription PCR (qRT-PCR) have become the most important and reliable tools to study differential gene expression in fish, where species-specific antibodies are scarce. Although qRT-PCR combines advantages of specificity, sensitivity, speed, throughput and reproducibility over conventional methods an accurate normalization of data is fully required [1]. Errors in the quantification of mRNA transcripts arise from any variation in the amount of starting material between samples. A common strategy to overcome this problem is to simultaneously amplify a non-regulated housekeeping gene with those targeted to allow quantitative normalization of the experimental cDNA inputs. However, it has also been demonstrated that expression levels of these genes may vary considerably depending on cell types, tissues, experimental treatments and even under different dis