Simultaneous quantitation of equine cytokine mRNAs using a multi-probe ribonuclease protection assay

A rapid multi-probe ribonuclease protection assay (RPA) was developed to quantitate equine-specific cytokine mRNA levels in activated equine monocyte-derived macrophages (EMDM) and equine peripheral blood mononuclear cells (EPBMC). Eleven template plasmids specific to 10 equine cytokine genes and the β-actin gene were generated from which radiolabeled anti-sense RNA probes were produced. The multi-probe RPA simultaneously quantitated mRNA levels of equine IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, IFN-γ, TGF-1β and TNF-α in EPBMC and EMDM with coefficients of variation as low as 0.03–0.08 (3–8%) when normalized to β-actin expression. This sensitive and rapid assay provides a valuable tool for studies of equine immune responses.