PCR was performed under mineral oil in a total volume of 100 ixl. The 20 I~ l of RT reaction mixture was brought to 2 mM MgClz, 50 mM KC1, 10 mM Tris-HC1 (pH 8.3), 0.2 mM dNTP, and 1.5 I~ M of each required primer. Amplification was carried out in a Perkin-Elmer Cetus 480 Thermal Cycler (Ueberlingen). Taq polymerase (2.5 units) was added to start the reaction when the reaction mixture had reached 94 C (hot start). The cDNA was amplified in 33 cycles with 1 min denaturation at 94 C, 1 min annealing at different temperatures, and 1 rain extension at 72 C. Following the 33 cycles a final extension was performed for 7 min at 72 C. For iNOS-and [3-actin amplification the annealing temperature was decreased during the first 8 cycles (2x at 69 C, 2x at 68 C, 2x at 67 C, 2x at 66 C) followed by 25 cycles at 65 C (touchdown protocol) to increase the specificity. The amplification with the chimeric primers st1 and st2 is described below. The PCR products were analyzed on nondenaturing, vertical 1.4% agarose gels in I x TBE, stained with ethidium bromide, and detected under UV light. Fluorescence of the products was detected relative to each other and to the marker fragments, using an imaging system (Herolab, Wiesloch).