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Open Access Highly Accessed Methodology article

Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis

Claudia M Espitia12, Weiguo Zhao12, Omar Saldarriaga12, Yaneth Osorio12, Lisa M Harrison3, Michael Cappello3, Bruno L Travi12 and Peter C Melby124*

Author Affiliations

1 Research Service, Department of Veterans Affairs Medical Center, South Texas Veterans Health Care System, 7400 Merton Minter, San Antonio, Texas, USA

2 Department of Medicine, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas, USA

3 Department of Pediatrics, Yale School of Medicine, P.O. Box 208064. New Haven, Connecticut, USA

4 Department of Microbiology and Immunology, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas, USA

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BMC Immunology 2010, 11:31  doi:10.1186/1471-2172-11-31

Published: 22 June 2010

Abstract

Background

The Syrian hamster, Mesocricetus auratus, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT) PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules.

Results

Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-γ, TNF-α, IL-10, IL-12p40, TGF-β, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-ΔΔCt) method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters.

Conclusions

The duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections.