Real-time polymerase chain reaction assays for leukocyte CD and cytokine mRNAs of the Eastern woodchuck (Marmota monax).

Real-time polymerase chain reaction (PCR) assays were developed for woodchuck leukocyte cluster of differentiation (CD) and cytokine mRNA expression. Plasmid DNA standards of each marker (CD3, CD4, CD8, IL-2, IFN-gamma, TNF-alpha, IL-4, IL-10), and RNA standards from mitogen-stimulated woodchuck peripheral blood mononuclear cells (PBMCs) were used to validate and optimize the assays for TaqMan 7700 and iCycler PCR instruments. The complementary DNAs (cDNAs) produced by reverse transcription (RT) of RNA were quantified by real-time PCR against the plasmid DNA standards (6-8 log range) with detection of as few as 10-50 copies of amplicon cDNA per reaction. Analysis of unstimulated and concanavalin A-stimulated woodchuck PBMC demonstrated increased CD and cytokine mRNA expression following mitogenic activation. A liver sample from a woodchuck hepatitis virus (WHV) infected woodchuck with histologically confirmed acute hepatitis had increased intrahepatic CD and cytokine mRNAs compared to liver from an uninfected control woodchuck. The real-time PCR assays were highly specific for the woodchuck markers in PBMC and liver samples and were equally applicable for use in alternate real-time PCR instrumentation. These assays will enable the high-throughput analyses of mRNA markers during WHV infection, and thereby facilitate continued modelling of the immunopathogenesis and immunotherapy of human hepatitis B virus (HBV) infection.