Figure 7. Effect of insulin-based culture supplement or serum on HIOMT and red opsin gene regulation

Real time RT-PCR analysis was conducted to assess the effects of serum or an insulin-based supplement in culture media on HIOMT and red opsin mRNA levels. E7 retinal cells were cultured for two days in either basal medium containing 0.5% bovine serum albumin (BSA) or medium supplemented with insulin-transferrin-selenium (ITS) or medium supplemented with newborn calf serum (NCS). Total RNA (about 1 μg) was reverse transcribed and cDNA aliquots (a tenth of the RT reaction) were analyzed successively by real time PCR with primer pairs specific for HIOMT, red opsin, and GAPDH. For both HIOMT PCR curves and red opsin PCR curves, the differences in critical cycle number observed between the different culture conditions were converted into fold increase over control culture in basal medium, after correction of the RNA load in each sample by the critical cycle number of GAPDH PCR (only marginal corrections were required). A first series of 5 duplicate cultures (A) and a second series of eight duplicate cultures (B) were analyzed. Bars represent means for n=10 (A) and n=16 (B); error bars represent the SEM.