Analysis of cytokine gene expression by qRT-PCR

Samples were obtained from oropharyngeal swabs of dogs (n = 6) before and after treatment with LTC, and expression of IL-8, MCP-1, IL-12p40, and IFNγ genes was determined via quantitative real time-(qRT)-PCR, using previously published primers [34, 35]. Briefly, cDNA was prepared by isolation of RNA followed by reverse transcription using a commercial kit (Qiagen, Germantown, MD) followed by amplification using SYBR™ green primers (Bio-Rad, Hercules, CA). Amplification was performed using a qPCR MX3000p system instrument (Agilent, Santa Clara, CA). All primers were validated to have an efficiency > 90% using stimulated and unstimulated healthy dog PBMC. pRT-PCR was used to quantify cytokine transcript levels as shown previously [24].