Quantitation of ovine cytokine mRNA by real-time RT–PCR

In this study we describe for the first time the dynamics of the expression of the cytokines, IL-1β, IL-12p40, TNFα in ovine dendritic cells and macrophages after LPS stimulation. Real time RT–PCR was used for the quantitation of these cytokines and IL-4 and IFNγ as well as two potential housekeeping genes (HKG), ATPase and GAPDH, in mRNAs from ovine leucocyte populations. Both dual-labelled probes (TAMRA/FAM) and SYBR Green assays were utilised, using a Corbett Research RotorGene and ABI 7700 machine. In order to quantitate each cytokine in our assays all CT values were compared to a standard curve generated using plasmid DNA containing the cytokine of interest. To validate our assays, concanavalin A-stimulated peripheral blood mononuclear cells (PBMCs) and LPS-stimulated monocyte-derived dendritic cells (MoDC) and monocyte-derived macrophages (MDMØ) were examined. We found that peak cytokine mRNA expression was between 3 and 6 h for the cytokines examined except for IL-12p40 where peak cytokine release was around 12 h post-stimulation in MDMØ and PBMCs. However, in MoDCs, peak IL-12p40 mRNA expression was observed within 3–6 h. We have identified a sensitive and reliable method for the identification of ovine cytokine mRNAs.