Semi-quantitative analysis of multiple cytokines in canine peripheral blood mononuclear cells zby a single tube RT-PCR

Cytokines play an important role in the regulation of the immune system, bu t low circulating levels in plasma make routine measurement a difficult tas k. A new methodology based on single tube RT-PCR has been developed to dete rmine the expression of multiple canine cytokines (TNF-alpha, IL-2, IFN-gam ma, IL-I 8, IL-4, IL-6 and IL-10) using primers and protocols designed allo w specific amplification of the mRNAs. The technique is performed in one tu be in two consecutive steps, a specific transcription of the mRNA of a give n cytokine and amplification of the corresponding gene by PCR. The technique was used to analyse the mRNA cytokine profile of peripheral b lood mononuclear cells (PBMCs) from healthy dogs using two approaches: (i) analysis of PBMC isolated ex vivo; (ii) analysis of PBMC after in vitro cul tures with or without the mitogen ConA. The samples were separated in agaro se gels and the intensity of ethidium bromide signals quantified using stan dard video imaging equipment. Results were interpreted as the ratio of cyto kine to GAPDH expression. The results obtained show that the method is easy to use and reproducible. Therefore, this method of monitoring the mRNA cyt okine expression might be an useful tool for understanding the immune respo nse in dogs. (C) 2001 Elsevier Science B.V. All rights reserved.