Different N-terminal isoforms of Oct-1 control expression of distinct sets of genes and their high level in cancer cells promotes pro-oncogenic processes

High level of Oct-1 isoforms in cells promotes pro-oncogenic processes Total RNA obtained from Namalwa cell line stably transfected by Oct-1 isoforms: Oct-1A, or Oct-1L or Oct-1X compared to untransfected control Namalwa cells. For each of the experimental conditions, total RNA was extracted from four cell lines using TRIzol Reagent (Invitrogen), followed by clean up on Ambion columns (Illumina Total-Prep RNA Amplification Kit, Ambion). Biotin-labelled cRNA targets were synthesized starting from 1.5 µg of total RNA. Double stranded cDNA synthesis was performed with Illumina TotalPrep RNA Amplification Kit (Ambion), and biotin-UTP-labelled antisense RNA was transcribed in vitro using Ambion’s Kit. All steps of the labelling were performed according to the manufacturer’s protocol. After purification, targets were diluted in hybridization buffer at a concentration of 240 ng/µl, and hybridization was allowed to proceed at 58°C for 20 h. For microarray analysis, the Illumina HumanHT-12 v4 Sentrix Expression BeadChip was used. Hybridization of labelled cRNA to the BeadChip, washing, and scanning were performed according to the Illumina BeadStation 500× manual. The array signal was developed via 10-min incubation with streptavidin-Cy3. The HumanHT-12 v4 Sentrix Expression BeadChip was washed and subsequently dried via centrifugation for 4 min at a setting of 275 xg. The arrays were scanned on the Illumina BeadArray reader, a confocal-type imaging system with 532 (Cy3) nm laser illumination.