Leutenegger C M, Mislin C N, Sigrist B, Ehrengruber M U, Hofmann-Lehmann R, Lutz H
Clinical Laboratory, Department of Internal Veterinary Medicine, University of Zurich, Switzerland.
Vet Immunol Immunopathol. 1999 Nov 30;71(3-4):291-305. doi: 10.1016/s0165-2427(99)00100-2.
We have developed real-time PCR systems to quantitate feline cytokine gene expression. The method is based on the cleavage of fluorescent dye-labelled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by a Sequence Detection System. The feline-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation of complementary DNA versus genomic DNA amplification products. Quantitative analysis of cytokine cDNA concentrations was performed in comparison to feline GAPDH. Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in the efficiencies of RNA extraction and reverse transcription. GAPDH mRNAs were readily detectable in cDNAs prepared from unstimulated feline peripheral blood mononuclear cells (PBMCs) and from frozen cell pellets, while cytokines (Interleukin (IL)-4, IL-10, IL-12 p35, IL-12 p40, IFNgamma, IL-16) were expressed at variable amounts. IFNgamma transcription was found to be upregulated in stimulated PBMCs and feline cell lines. The synthesis of cDNA and the performance of the PCR in separate tubes proved to be of superior sensitivity compared to a single-tube based system. The assays described are highly reproducible, require no post-PCR manipulation of the amplicons and permit the analysis of several hundred PCR reactions per day. With this method it is possible to detect and quantify cytokine mRNA expression reliably in small amounts of cells even after storage of samples for at least 5 years.
我们开发了实时PCR系统来定量猫细胞因子基因的表达。该方法基于在PCR过程中Taq DNA聚合酶的5'-3'核酸外切酶活性对荧光染料标记探针的切割，并通过序列检测系统测量荧光强度。猫特异性TaqMan探针被设计为包含一个内含子，从而能够区分互补DNA与基因组DNA扩增产物。与猫GAPDH相比，对细胞因子cDNA浓度进行了定量分析。来自普遍表达的管家基因GAPDH的信使RNA（mRNA）被证明可作为扩增对照，并可校正RNA提取和逆转录效率的差异。在从未刺激的猫外周血单核细胞（PBMC）和冷冻细胞沉淀制备的cDNA中很容易检测到GAPDH mRNA，而细胞因子（白细胞介素（IL）-4、IL-10、IL-12 p35、IL-12 p40、IFNγ、IL-16）的表达量各不相同。发现IFNγ转录在刺激的PBMC和猫细胞系中上调。与基于单管的系统相比，在单独的管中合成cDNA和进行PCR被证明具有更高的灵敏度。所描述的检测方法具有高度可重复性，无需对扩增子进行PCR后处理，并且每天可分析数百个PCR反应。使用这种方法，即使在样品储存至少5年后，也能够在少量细胞中可靠地检测和定量细胞因子mRNA的表达。
