Real Time-PCR and ChIP validation of differential regulation of selected transcripts. (A) Correlation between Microarray and Real Time-PCR data. 9 transcripts identified as differentially expressed by microarray (TXNRD1, IGFBP3, P2RY2, Cyp26B1, SEMA3B, SEMA3F, VAV3, AKAP12 and APCDD1) were examined by Real Time-PCR for 1,25(OH)2D-induced changes in expression at the 6, 24, and 48 h. Fold changes of RT-PCR validated transcripts were compared across their fold change identified in microarray analysis. The regression line was defined by the following equation: PCR fold change = 0.91(Microarray data fold change) + 0.09; r2 = 0.64. (B) ChIP assays of VDR recruitment to putative VDR binding sites. RWPE1 cells were treated with vehicle or 10 nM 1,25(OH)2D for 3 h. DNA precipitates were measured with RT-PCR using primers spanning known VDREs (CYP24, TRPV6 and SEMA3B) and predicted VDREs (CYP26B1 and AKAP12). The results are shown as mean ± SEM (n = 3).
Kovalenko et al. BMC Genomics 2010 11:26 doi:10.1186/1471-2164-11-26