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

Time-resolved fluorometry was applied in the detection of RT-PCR amplified mRNAs for the Th1 and Th2 cell-derived cytokines interferon gamma (IFN-gamm a) and interleukin (IL-)4, respectively, RNA from stimulated cells was reve rse transcribed and the cDNAs for the cytokine mRNAs and the constantly exp ressed beta-actin (beta-ACT) mRNA were simultaneously amplified in one mult iplex PCR reaction. The PCR conditions were optimized to minimize mutual in hibition of individual amplifications. One of the PCR primers in each prime r pair was biotinylated, and the PCR products were captured onto streptavid in-coated microtitre plates. The three PCR products were detected with thre e different lanthanide labelled target-specific probes in solution hybridiz ation. IFN-gamma, IL-4 and beta-ACT were detected with europium (Eu), terbi um (Tb) and samarium (Sm) labelled probes, respectively, using time-resolve d flurometry, Small cell numbers used in microtitre plate cultures were suf ficient to detect cytokine messages after mitogen stimulation. This sequenc e-based method provides a sensitive, specific, fast and nonisotopic alterna tive to conventional blotting and hybridisation with radioactive probes. In addition, the multiplex fluorogenic dye detection facilitates relative qua ntification of target mRNAs, (C) 1999 Academic Press.