A quantitative competitive reverse transcription-polymerase chain reaction technique to measure porcine interferon-gamma.
In swine, as in other mammals, interferon-gamma (IFN-gamma) plays an important role in the regulation of the immune response. The objective of this work was to develop a sensitive, quantitative competitive reverse transcription-polymerase chain reaction (qcRT-PCR) assay, using an internal cRNA standard, to monitor the expression of porcine IFN-gamma gene. We applied this qcRT-PCR assay to quantify the expression of IFN-gamma transcripts in peripheral blood mononuclear cells (PBMC) incubated for different times with or without concanavalin A (ConA). Results showed that the expression of the IFN-gamma transcripts in PBMC occurred within the first 2 h after mitogenic stimulation and reached a peak after 24 h of incubation with ConA. The qcRT-PCR technique provides an efficient and sensitive tool for studying the expression of porcine IFN-gamma gene under a variety of experimental conditions. This will help to understand the regulation of T cell-mediated immune responses by IFN-gamma at the gene level.
Lessard
M
M
Agriculture and Agri-Food Canada, Dairy and Swine Research and Development Centre, Lennoxville, Quebec.
He
S
S
Benkel
B
B
D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D007371:Interferon-gamma / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology*; D008213:Lymphocyte Activation; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012333:RNA, Messenger / Q000032:analysis; D013552:Swine / Q000097:blood / Q000235:genetics / Q000276:immunology*; D014158:Transcription, Genetic
D017931:DNA Primers; D012333:RNA, Messenger; D003208:Concanavalin A; D007371:Interferon-gamma