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 beta-actin gene were generated from which radiolabeled anti-sense RNA probes were produced. The multi-probe RPA simultaneously quantitated mRNA levels of equine IL-1alpha, IL-1beta, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, IFN-gamma, TGF-1beta and TNF-alpha in EPBMC and EMDM with coefficients of variation as low as 0.03-0.08 (3-8%) when normalized to beta-actin expression. This sensitive and rapid assay provides a valuable tool for studies of equine immune responses.
Lim
W-S
WS
Department of Veterinary Pathobiology, Texas A&M University, MS 4467, College Station, TX 77843-4467, USA.
Edwards
J F
JF
Boyd
N K
NK
Payne
S L
SL
Ball
J M
JM
D000818:Animals; D016207:Cytokines / Q000235:genetics*; D006736:Horses; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D015336:Molecular Probe Techniques; D012333:RNA, Messenger / Q000032:analysis*; D012260:Ribonucleases / Q000494:pharmacology*
D016207:Cytokines; D012333:RNA, Messenger; D012260:Ribonucleases