Reverse transcription real-time PCR for detection of porcine interferon α and β genes

Abstract

A few studies provided convincing evidence of constitutive expression of type I interferons (IFNs) in humans and mice, and of the steady-state role of these cytokines under health conditions. These results were later confirmed in pigs, too. In line with this tenet, low levels of IFN-α/β can be detected in swine tissues in the absence of any specific inducer. These studies are compounded by the utmost complexity of type I IFNs (including among others 17 IFN-α genes in pigs), which demands proper research tools. This prompted us to analyse the available protocols and to develop a relevant, robust, reverse transcription (RT) real-time polymerase chain reaction (PCR) detection system for the amplification of porcine IFN-α/β genes. The adopted test procedure is user-friendly and provides the complete panel of gene expression of one subject in a microtitre plate. Also, a proper use of PCR fluorochromes (SYBR(®) versus EvaGreen(®) supermix) enables users to adopt proper test protocols in case of low-expression porcine IFN-α genes. This is accounted for by the much higher sensitivity of the test protocol with EvaGreen(®) supermix. Interestingly, IFN-β showed the highest frequency of constitutive expression, in agreement with its definition of 'immediate early' gene in both humans and mice. Results indicate that the outlined procedure can detect both constitutively expressed and virus-induced IFN-α/β genes, as well as the impact of environmental, non-infectious stressors on the previous profile of constitutive expression.