Quantitative Reverse Transcription PCR

RNA was extracted from 2D and dissociated 3D cultures using an RNeasy Plus kit (Qiagen), and 1 μg of total RNA was reverse transcribed with the iScript™ first-strand cDNA synthesis kit (BioRad). Following cDNA synthesis, quantitative PCR was set up using SsoFast™ EvaGreen^® supermix (Biorad) with optimized cycling conditions for LightCycler 480II (Roche). Based on preliminary analysis of microarray data, the following genes were selected as being differentially up- or downregulated between 2D and 3D conditions: Jun proto-oncogene (JUN), sirtuin 2 (SIRT2), Ras-related GTP binding protein (RAB6A), and CDC-like kinase 1 (CLK1), MYC, ADAM metallopeptidase with thrombospondin type 1 motif 6 (ADAMTS6), and BMI polycomb ring finger oncogene (BMI1). Housekeeping genes used were glucuronidase beta (GUSB), heat shock protein 90 kDa alpha class B member 1 (HSP90AB1), and hypoxanthine phosphoribosyltransferase 1 (HPRT1).