Isolation of RNA and reverse transcription (RT)

High pure total-RNA isolation kit (Roche, Basel, Switzerland) was used for isolation of cellular RNA. The medium was removed and samples were washed twice with 5 ml of ice-cold PBS. Cells were scraped off, transferred to clean tubes and centrifuged at 20 800 × g for 5 min at 4°C. After that, lysis buffer was added and RNA isolation was carried out according to manufacturer's instructions. Similarly, RNA from biopsies was isolated using the same kit. Isolated RNA was used for cDNA synthesis. RNA (500 ng) was transcribed using Transcriptor First Strand cDNA Synthesis Kit (Roche) according to manufacturer's instructions. Prepared cDNA (20 μl) was diluted with RNase-free water to a total volume of 100 μl and 5 μl of this solution was employed for qRT-PCR and microarrays.