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首页> 外文期刊>Cytokine >Simultaneous detection of IFN-gamma and IL-4 mRNAs using RT-PCR and time-resolved fluorometry.
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Simultaneous detection of IFN-gamma and IL-4 mRNAs using RT-PCR and time-resolved fluorometry.

机译：使用RT-PCR和时间分辨荧光法同时检测IFN-γ和IL-4 mRNA。

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Time-resolved fluorometry was applied in the detection of RT-PCR amplified mRNAs for the Th1 and Th2 cell-derived cytokines interferon gamma (IFN-gamma) and interleukin (IL-)4, respectively. RNA stimulated cells was reverse transcribed and the cDNAs for the cytokine mRNAs and the constantly expressed beta-actin (beta-ACT) mRNA were simultaneously amplified in one multiplex PCR reaction. The PCR conditions were optimized to minimize mutual inhibition of individual amplifications. One of the PCR primers in each primer pair was biotinylated, and the PCR products were captured onto streptavidin-coated microtitre plates. The three PCR products were detected with three different lanthanide labelled target-specific probes in solution hybridization. IFN-gamma, IL-4 and beta-ACT were detected with europium (Eu), terbium (Tb) and samarium (Sm) labelled probes, respectively, using time-resolved fluorometry. Small cell numbers used in microtitre plate cultures were sufficient to detect cytokine messages after mitogen stimulation. This sequence-based method provides a sensitive, specific, fast and nonisotopic alternative to conventional blotting and hybridisation with radioactive probes. In addition, the multiplex fluorogenic dye detection facilitates relative quantification of target mRNAs. Copyright 1999 Academic Press.
机译：时间分辨荧光分析法分别用于检测Th1和Th2细胞源性细胞因子干扰素γ（IFN-γ）和白介素（IL-）4的RT-PCR扩增mRNA。 RNA刺激的细胞被反转录，并且在一个多重PCR反应中同时扩增了细胞因子mRNA和恒定表达的β-肌动蛋白（β-ACT）mRNA的cDNA。优化了PCR条件，以最大程度地减少对单个扩增的相互抑制。每个引物对中的一个PCR引物被生物素化，并将PCR产物捕获到链霉亲和素包被的微量滴定板上。在溶液杂交中，用三种不同的镧系元素标记的靶标特异性探针检测了三种PCR产物。使用时间分辨荧光法分别用labeled（Eu），ter（Tb）和sa（Sm）标记的探针检测IFN-γ，IL-4和β-ACT。在微量滴定板培养中使用的小细胞数量足以检测促细胞分裂剂刺激后的细胞因子信息。这种基于序列的方法为常规印迹和放射性探针杂交提供了灵敏，特异，快速且非同位素的替代方法。另外，多重荧光染料检测促进了靶mRNA的相对定量。版权所有1999，学术出版社。

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