RNA Extraction and qRT-PCR for MACC1 and IGFBP2

RNA isolation was performed using the Universal RNA extraction kit (Roboklon, Berlin, Germany). A quantity of 50 ng of total RNA was used for MuMLV-based cDNA synthesis (Biozym, Vienna, Austria). Primers for MACC1 were described elsewhere [1 (link)]. The primers for human IGFBP2 are forward: 5′-GCC CTC TGG AGC ACC TCT ACT-3′ and reverse: 5′-CAT CTT GCA CTG TTT GAG GTT GTA C-3′. All primers were synthesized at BioTeZ Berlin-Buch. qRT-PCR was performed as described earlier [3 (link)]. In brief, after initial denaturation at 95 °C, the amplification was performed for 40 cycles of denaturation (5 s; 95 °C) and a combined primer annealing and elongation step (45 s; 60 °C). Data were analyzed with LightCycler 480 Software release 1.5.0 SP3 (Roche Diagnostics, Penzberg, Germany). Mean values were calculated from duplicates. Each mean value of the expressed gene was normalized to its G6PDH level.