Relative gene expression levels of E2F3 and NM_017774 in various cancer cell lines with and without 6p22.3 amplification. Both genes are markedly upregulated in amplified cell lines. Cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown under standard cell culturing conditions. RNA isolations were carried out according to the manufacturer's specifications using DNase I system in combination with the RNeasy kit (Qiagen, Hilden, Germany). RNA concentrations were determined with a spectrophotometer. For each cell line, 250 ng total RNA was used as starting material for complementary DNA (cDNA) synthesis combined with Oligo-dT (Roche, Basel, Switzerland) as primer. Real-time PCR was performed in duplicates in 20 μl reactions containing: 2 μl cDNA template (from 1:2 dilutions of cDNA synthesis reaction), 10 μl FastStart SYBR Green I PCR Master Mix (Roche), MgCl2 as well as forward and reverse primer mix (10 mM each). Thermal cycling conditions for the LightCycler Instrument (Roche) were: one cycle at 95°C for 10 min at steps of 20°C/s (activation), 40 cycles at 95°C for 15 s at 20°C/s, 55°C for 10 s at 20°C/s and 72°C for 10 s at 5°C/s (amplification) and one additional cycle at 95°C for 1 s at 20°C/s, 65°C for 15 s at 20°C/s and 99°C for 1 s at 0.05°C/s (melting). Relative levels of expression were determined using the method as described by Livak and Schmittgen (2001). All samples were normalized against glyceraldehydes-3-phosphate dehydrogenase (G3PDH).