FIG. 3.

Relative expression of ERK1c and ERK1 in MCF7 and HeLa cells. (A) Real-time PCR was performed in a LightCycler (Roche) with the FastStart DNA Master Hybridization Probes kit (Roche), following the manufacturer's instructions for the composition of the reaction mixture. The fluorescent hybridization probes for sequence-specific detection (synthesized by Tib Molbiol) were used. Real-time PCR was performed by a touchdown procedure (stepwise decrease of the annealing temperature for the amplification primers). To produce the cDNA, total RNA was reverse transcribed using the first-strand cDNA synthesis kit for RT-PCR (Roche) with random hexamers as primers, according to the manufacturer's recommendations. (B) To generate standard curves, template dilution series (cDNA mixture in four subsequent 1:50 dilution steps) were added to the reaction mixture. Results were processed using the LightCycler software package (version 3.5.28). The crossing points (CP) are indicated.