Relative quantification of POU5F1 and NANOG. The importance of proper reference gene selection is illustrated by comparing normalized data of the pluripotency factors Oct4 and Nanog to different reference sets. The Cq values for the pluripotency genes POU5F1 and NANOG were both normalized following the ΔΔCt-method (applying qbasePLUS software), against the geometric average of two different reference sets: on the one hand for ACTB, GAPDH and PPIA (blue triangle); on the other hand for B2M, RPL13A and Alu repeats (green rhomb). The resulting values represent the fold change in expression levels of the pluripotency factors, and are displayed for POU5F1 and NANOG for both normalization methods in respectively panel A and B. Data shown are obtained from differentiation Experiment 2 (sample isolation every 24 h during 6 days of differentiation).
Vossaert et al. BMC Molecular Biology 2013 14:21 doi:10.1186/1471-2199-14-21