Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis.
Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis.
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DOI：
10.1186/1471-2172-11-31
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发表时间：
2010-06-22
期刊：
影响因子：
3
通讯作者：
Melby PC
中科院分区：
文献类型：
--
作者：
Espitia CM;Zhao W;Saldarriaga O;Osorio Y;Harrison LM;Cappello M;Travi BL;Melby PC
叙利亚仓鼠，auratus Mesocricetus，具有独特的免疫学特征，对细胞内病原体非常敏感。在仓鼠身上的研究受限于相对缺乏进行免疫学研究的工具。为了解决这一限制，我们开发了双实时逆转录酶（RT） PCR检测，用于相对定量仓鼠细胞因子、趋化因子和相关免疫反应分子的mrna。合成Real-time RT-PCR引物和探针，分析白细胞介素(IL)-4、IFN-γ、TNF-α、IL-10、IL-12p40、TGF-β、IL-13、IL-21、趋化因子配体（CCL） 22、CCL17、趋化因子（C-C motif）受体4和FoxP3的表达。对每个实时RT-PCR检测进行标准曲线和验证实验，允许我们使用比较Ct （2-ΔΔCt）方法来计算基因表达的变化。通过比较未感染和感染巴拿马利什曼原虫的仓鼠皮肤和淋巴结组织中mRNA的表达，验证了实时RT - PCR技术在生物模型中的应用。双工实时RT - PCR检测为仓鼠细胞因子转录的定量提供了一种强有力的方法，它们在皮肤利什曼病模型中的应用表明，平衡的1型和2型细胞因子反应有助于慢性、非进展性疾病的病程。这些新的分子工具将进一步促进对仓鼠疾病机制的研究，不仅对利什曼病模型，而且对其他病毒、细菌、真菌和寄生虫感染模型。
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. 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. 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.
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影响因子：
5.8
作者：
Bilate, AMB;Salemi, VMC;Cunha-Neto, E
通讯作者：
Cunha-Neto, E
影响因子：
6.4
作者：
Paessler, S;Aguilar, P;Weaver, SC
通讯作者：
Weaver, SC
影响因子：
4.9
作者：
Iñiguez, SR;Dea-Ayuela, MA;Bolas-Fernández, F
通讯作者：
Bolas-Fernández, F
影响因子：
3.1
作者：
Melby, PC;Tryon, VV;Freeman, GL
通讯作者：
Freeman, GL
影响因子：
1.8
作者：
Requena, JM;Soto, M;Alonso, C
通讯作者：
Alonso, C