Simultaneous detection of IFN-γ and IL-4 mRNAS using RT-PCR and time-resolved fluorometry

被引:16
作者
Halminen, M
Sjöroos, M
Mäkelä, MJ
Waris, M
Terho, E
Lövgren, T
Ilonen, J
机构
[1] Univ Turku, Dept Virol, FIN-20520 Turku, Finland
[2] Univ Turku, Dept Biotechnol, FIN-20520 Turku, Finland
[3] Univ Turku, Dept Paediat, FIN-20520 Turku, Finland
[4] Univ Turku, Dept Pulm Dis & Clin Allergol, FIN-20520 Turku, Finland
[5] Univ Turku, Turku Immunol Ctr, FIN-20520 Turku, Finland
基金
芬兰科学院;
关键词
cytokine; IFN-gamma; IL-4; mRNA quantification; time-resolved fluorometry;
D O I
10.1006/cyto.1998.0392
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 from 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 flurometry, 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, (C) 1999 Academic Press.
引用
收藏
页码:87 / 93
页数:7
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