Selection of reference genes suitable for normalisation of IFN-γ and TNF mRNA expression by real-time SYBR Green polymerase chain reaction in porcine duodenum tissue

DOI: 10.5176/2382-5685_VETSCI14.34

Authors: Stephen C. Mansbridge, Alexander M. Mackenzie, Vasil Pirgozliev, Louise S. Skinner, Naomi Evans and Alan H. Stewart and Carrie L. Walk

Abstract:

The quantification of cytokine gene expression has become an invaluable tool in veterinary science to characterise immune responses to a variety of antigens. Six candidate reference genes for normalising duodenal cytokine gene expression data obtained through reverse-transcription polymerase chain reaction (RT-qPCR) with SYBR® green dye were assessed for their suitability using the qBase+ framework. Their stability was then validated using IFN-γ and TNF cytokine gene expression data from a total of 32 samples taken from pigs three weeks post weaning. The study concluded that the most appropriate combination of reference genes to normalise cytokine data in the duodenum of pigs three weeks post weaning were GSR and GPI, with ACTB being the least stable.

Keywords: geNorm, cytokine expression, RT-qPCR