Figueiredo M D, Salter C E, Andrietti A L P, Vandenplas M L, Hurley D J, Moore J N
Department of Large Animal Medicine, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7385, USA.
Vet Immunol Immunopathol. 2009 Sep 15;131(1-2):65-72. doi: 10.1016/j.vetimm.2009.03.013. Epub 2009 Mar 27.
Quantification of gene expression using real-time reverse transcription quantitative PCR (RT-qPCR) is a reliable method to monitor cellular responses to pro-inflammatory stimuli. The main objective of this study was to validate a set of equine primer pairs that can be routinely used to monitor expression of genes that are central to inflammatory and immune responses. This paper describes the steps used to optimize and validate 29 equine primer pairs for RT-qPCR assays using SYBR Green detection. To validate these assays, monocytes were isolated from three horses and stimulated with Escherichia coli LPS. Because four of the 29 genes demonstrated poor amplification efficiency due to weak induction of gene expression by LPS, monocytes were stimulated with alternative agents (PMA and Poly I:C) known to induce gene expression in monocytes. These agents, acting through different pathways than LPS, improved the level of gene expression and yielded good amplification efficiency for these genes. PCR efficiency was based on a standard curve for each gene and the calculated efficiency was approximately 100% for all 29 genes. The PCR efficiencies for the majority of the target genes were equivalent to that of the housekeeping gene (18S rRNA) used in all experiments. Dissociation curve analysis and gel electrophoresis revealed a single product for each gene analyzed. To exemplify utilization of the validated primer pairs in studies of inflammatory cell activation, temporal changes in mRNA expression of a subset of genes were monitored in equine monocytes incubated with LPS. The availability of the 29 validated primer pairs reported herein will allow investigators to elucidate the response of horses to a variety of inflammatory stimuli and will further our understanding of disease pathogenesis in horses.
使用实时逆转录定量聚合酶链反应（RT-qPCR）对基因表达进行定量分析是监测细胞对促炎刺激反应的可靠方法。本研究的主要目的是验证一组马源引物对，这些引物对可常规用于监测对炎症和免疫反应至关重要的基因的表达。本文描述了使用SYBR Green检测优化和验证用于RT-qPCR分析的29对马源引物对的步骤。为了验证这些分析方法，从三匹马中分离出单核细胞，并用大肠杆菌脂多糖（LPS）进行刺激。由于29个基因中的4个基因由于LPS对基因表达的诱导较弱而显示出较差的扩增效率，因此用已知可诱导单核细胞基因表达的替代试剂（佛波酯和聚肌胞苷酸）刺激单核细胞。这些试剂通过与LPS不同的途径起作用，提高了基因表达水平，并为这些基因产生了良好的扩增效率。PCR效率基于每个基因的标准曲线，所有29个基因的计算效率约为100%。大多数靶基因的PCR效率与所有实验中使用的管家基因（18S rRNA）相当。解离曲线分析和凝胶电泳显示每个分析的基因都有单一产物。为了举例说明经验证的引物对在炎症细胞激活研究中的应用，在与LPS孵育的马单核细胞中监测了一部分基因的mRNA表达的时间变化。本文报道的29对经验证的引物对的可用性将使研究人员能够阐明马对各种炎症刺激的反应，并将进一步加深我们对马疾病发病机制的理解。
