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

Claudia M. Espitia, Weiguo Zhao, Omar Saldarriaga, Yaneth Osorio, Lisa M. Harrison, Michael Cappello, Bruno Travi, Peter Melby

Research output: Contribution to journalArticle

19 Citations (Scopus)

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.

Original languageEnglish (US)
Article number31
JournalBMC Immunology
Volume11
DOIs
StatePublished - Jun 22 2010
Externally publishedYes

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Cutaneous Leishmaniasis
Reverse Transcriptase Polymerase Chain Reaction
Cricetinae
Real-Time Polymerase Chain Reaction
Cytokines
Messenger RNA
Mesocricetus
Chemokines
CCR Receptors
Biological Models
Parasitic Diseases
Leishmaniasis
Interleukin-13
Mycoses
Interleukins
Leishmania
Virus Diseases
Bacterial Infections
Interleukin-4
Interleukin-10

ASJC Scopus subject areas

  • Immunology

Cite this

Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis. / Espitia, Claudia M.; Zhao, Weiguo; Saldarriaga, Omar; Osorio, Yaneth; Harrison, Lisa M.; Cappello, Michael; Travi, Bruno; Melby, Peter.

In: BMC Immunology, Vol. 11, 31, 22.06.2010.

Research output: Contribution to journalArticle

Espitia, Claudia M. ; Zhao, Weiguo ; Saldarriaga, Omar ; Osorio, Yaneth ; Harrison, Lisa M. ; Cappello, Michael ; Travi, Bruno ; Melby, Peter. / Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis. In: BMC Immunology. 2010 ; Vol. 11.
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