Differential chemokine expression patterns in tonsillar disease

Mandapathil, M.; H.Beier, U.; Graefe, H.; Kröger, B.; Hedderich, J.; Maune, S.; Meyer, J. E

Reverse transcribed cDNA products (0.2 5µl) were incubated in 50 µl reaction mixture containing 0.2 µM 5'-3' sequence specific sense oligonucleotide primers, 0.2 µM of 3'-5' corresponding antisense oligonucleotide primers, 200 µM dNTP's, 1.5 mM MgCl 2 , 5.0 µl 10x PCR-buffer, and 2.5 U Taq-polymerase (Gibco, Ingolstadt, Germany). The reaction mixture was covered with a mineral oil layer (Applied Biosystem, Weiterstadt, Germany) to prevent evaporation. The PCR was conducted in a Biometra T 3 thermocycler. Following initial denaturation (3 min at 95°C), high stringency PCR was run for 34 cycles (94°C for 75 sec, 60°C for 30 sec, and 72°C for 2 min) with an increased annealing temperature of 67°C in the first two cycles, to amplify the RANTES, Eotaxin-1, Eotaxin-2 and G3PDH cDNA. In case of the other chemokines, the PCR parameters were modified to a 40 cycles of 95°C for 60 sec, 60°C for 30 sec, and 72°C for 2 min, with an increased annealing temperature of 68° to 60° over the first 8 cycles. After PCR, all samples were subjected to ethidium-bromide stained 1.5% agarose gel electrophoresis.