Normalisation of EphB4 expression to housekeeping genes PBGD and HPRT1. EphB4 expression normalised to PBGD in prostate cancer cell lines LNCaP, DU145 and PC3. (A) Triplicate amplifications of EphB4 and PBGD in three samples for each cell line were analysed to determine the relative levels of EphB4 expression using the BioRad iCycler. All EphB4/PBGD ratios were close to 1 indicating that there are comparable amounts of both templates in each RNA samples. (B) Dilutions of a single RNA sample for each cell line were also amplified in triplicate with both gene primer sets to confirm that the ratio was consistent regardless of starting template concentration. (C) Triplicate amplifications of EphB4, PBGD and HPRT1 in three samples for each cell line using the Corbett Rotorgene confirmed the previous result for EphB4/PBGD and showed a similar result when EphB4 expression was normalised to a second housekeeping gene.