Quantitation of equine cytokine mRNA expression by reverse transcription-competitive polymerase chain reaction

A reverse transcription-competitive polymerase chain reaction (RT-cPCR) method was developed to quantitate equine interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p35, IL-12 p40, interferon-gamma (INF-gamma), tumor necrosis factor-alpha (TNF-alpha), and beta-actin mRNA expression. Using primers based on equine-specific sequences, these cytokines could be detected in concanavalin A-stimulated peripheral blood mononuclear cells. The specificity of the amplified product was confirmed by sequencing. For each cytokine, the assay was made quantitative by generating competitor DNA fragments (mimic) containing the same primer template as a equine cytokine, but differing in size to make them distinguishable on an agarose gel. Serial dilutions of the mimic were added to PCR reactions containing constant amount of equine cDNA. Following gel electrophoresis and ethidium bromide staining, densitometric analysis of the bands corresponding to the target and mimic were used to construct a standard curve from which the amount of target cDNA was derived. Quantitation of IL-6 gene expression from a cDNA sample on four different days gave a coefficient of variation or 6.6%. Sample-to-sample variation in the efficiency of the reverse transcription as well as in the quantity of quality of starting RNA was considerably attenuated by normalizing the results to beta-actin mRNA expression used as a house-keeping gene. Small differences (2-fold) in cytokine mRNA expression were reliably detected. The sensitivity and reproducibility of this technique will make it valuable in following changes in equine cytokine gene expression in vitro and in vivo. In addition, the RT-cPCR technique described will have broad applicability for quantitation of cytokine gene expression in other animal species of veterinary interest.