Reference Gene Selection Of QRT-PCR And Effects On ASAH1Expression Level In PRRSV Infected Bone Marrow

Aims:Reference gene is used as an endogenous control in RT-PCR. It plays an important in standardizing the target gene. But the expression levels may be changed after infected by the PRRSV. Thus it is necessary to choose a stable expression reference gene. The digital gene expression profile (DGE) was conducted in our lab. The data after bioinformatics analysis showed that acid ceramidase (ASAH1) significantly changed. This study investigated the interaction between ASAH1and host during the infection process of PRRSV. In addition it provided perspective to understand the mechanism of PRRSV infection.Methods:nine reference genes was detected by RT-PCR and their expression stability were evaluated by geNorm software. The expression levels of ASAH1gene and protein were examined by RT-PCR and western blotting respectively.Results:In the marrow after the PRRSV infection, β-actin is the most stable reference gene and the B2m is the most unstable reference gene.β-actin>GAPDH>SDHA>5S>18S>TOP2B>HPRT>PKGl>Bm2.In digital expression profile (DGE) the ASAH1gene is significantly increased. The RT-PCR and western blotting show that ASAH1remarkable increase both in gene and protein levels.Conclusion:β-actin is the most stable reference gene in marrow tissue after PRRSV infection. The ceramide, the substrate of ASAH1, is the key molecule in sphingomyelin pathway signal system. ASAH1regulates the NF-κ b signal pathway by hydrolyzing ceramide. The increased expression of ASAH1may play an vital role in apoptosis and inflammatory reaction. But the mechanism needs to be investigated.