Quantitation of ovine cytokine mRNA by real-time RT-PCR.
In this study we describe for the first time the dynamics of the expression of the cytokines, IL-1beta, IL-12p40, TNFalpha in ovine dendritic cells and macrophages after LPS stimulation. Real time RT-PCR was used for the quantitation of these cytokines and IL-4 and IFNgamma as well as two potential housekeeping genes (HKG), ATPase and GAPDH, in mRNAs from ovine leucocyte populations. Both dual-labelled probes (TAMRA/FAM) and SYBR Green assays were utilised, using a Corbett Research RotorGene and ABI 7700 machine. In order to quantitate each cytokine in our assays all C(T) values were compared to a standard curve generated using plasmid DNA containing the cytokine of interest. To validate our assays, concanavalin A-stimulated peripheral blood mononuclear cells (PBMCs) and LPS-stimulated monocyte-derived dendritic cells (MoDC) and monocyte-derived macrophages (MDMØ) were examined. We found that peak cytokine mRNA expression was between 3 and 6 h for the cytokines examined except for IL-12p40 where peak cytokine release was around 12 h post-stimulation in MDMØ and PBMCs. However, in MoDCs, peak IL-12p40 mRNA expression was observed within 3-6 h. We have identified a sensitive and reliable method for the identification of ovine cytokine mRNAs.
Budhia
Sangeeta
S
Department of Veterinary Medicine, University of Cambridge, Cambridge, CB3 0ES, United Kingdom.
Haring
Lisa F
LF
McConnell
Ian
I
Blacklaws
Barbara A
BA
D000251:Adenosine Triphosphatases / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics; D018664:Interleukin-12 / Q000096:biosynthesis / Q000235:genetics; D053762:Interleukin-12 Subunit p40; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology; D008297:Male; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012756:Sheep / Q000235:genetics* / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics
D016207:Cytokines; D018076:DNA, Complementary; D007375:Interleukin-1; D053762:Interleukin-12 Subunit p40; D008070:Lipopolysaccharides; D021122:Protein Subunits; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D000251:Adenosine Triphosphatases