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Real time RT-PCR for porcine cytokines to assist vaccine efficacy evaluation

Real time RT-PCR cytokine assays with samples gained from vaccinated and challenged swine has been developed. One assay established allows the simultaneous detection of 9 sequences. 3 different sets of 3 targets each are measured in a triplex-format using TaqMan-probes. Six targets can be selected from the following lymphokines: IL-2, IL-4, IL-5, IL-10, and IFN-gamma, and have the proinflammatory cytokines IL-1alpha and IL-6.

Simultaneously the most suitable combination of 3 out of the four housekeeping genes (beta-actin, HPRT, GAPDH, and cyclophilin) can be selected, and their averaged expression values constitute a normalisation factor. The raw data of all targets of interest is than calculated relative to this normalisation factor, making also eventual changes of the relative expression level of the single housekeeping genes controllable, and quantifiable. Performance and suitability for the real time PCR was controlled.

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