Validation of microarray results by quantitative real time PCR Fi

Validation of microarray results by quantitative real time PCR Five genes involved in the following website presentation antigen class I pathway were studied by qRT PCR SLA Ia, TAP1, TAP2, PSMB8 and PSMB9. PPIA, down regulated during infec tion, and TNF, even if not detected as differentially expressed in our transcriptome Inhibitors,Modulators,Libraries experiment, were also cho sen for validation. qRT PCR were performed for a subset of conditions Inhibitors,Modulators,Libraries at 0, 2, 4, 8, and 12 h pi. We confirmed that SLA Ia genes were down regulated during infection from 8 h pi. We also observed a clear down regulation of TAP1 and TAP2 from 8 and 4 h pi, respectively. An early down regulation of PSMB8 and PSMB9 was detected before 2 h pi. TNF was strongly up regulated from 4 h pi and PPIA was down reg ulated from 2 h pi.

Cell surface expression of MHC class I and MHC class II molecules on PK15 cells during PrV infection Since Inhibitors,Modulators,Libraries our experiments, as well as other studies, have clearly indicated a down Inhibitors,Modulators,Libraries regulation of the MHC class I genes during PrV infection, we checked, by flow cytome try, for a down regulation of surface MHC class I mole cules on PrV infected PK15 cells at 8 h pi. To visualize infected cells, we used, in the same experimental condi tions, a recombinant PrV strain expressing the green fluorescent protein. Ninety percent of the cells appeared infected and 73% of these infected cells expressed surface MHC class I molecules on their surface while 89. 1% and 83. 9% of the mock infected cells expressed MHC class I molecules at 0 and 8 h pi, respectively. The MHC class I mean flu orescence intensity of Inhibitors,Modulators,Libraries infected cells at 8 h pi was 50.

9% of that of mock infected cells thus confirming a clear decrease of MHC class I molecules expression on the surface of infected cells. As a control, this research we observed that the expression of tubulin, detected by Western blot, remains unchanged even 8 h pi in PK15 cells. Since a significant variation in MHC class II transcript lev els during infection was detected in our transcriptome analysis, we also analyzed the expression of MHC class II molecules on the surface of PK15 cells. Our results show that 5. 5% of mock infected cells and infected cells expressed surface MHC class II mol ecules. However, we could not detect any differential expression between infected and mock infected cells at 8 h pi. Discussion A joint PrV porcine epithelial cell transcriptomic approach This work is the first study of PrV transcriptome expres sion during the time course of infection. Moreover, it is the first time that the gene expressions of both PrV and porcine cells during infection are analyzed simultaneously and we demonstrate that virus and host cell transcriptome modifications can be examined with a unique microarray combining viral and host cell probe sets.

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