Journal: BMC Cancer
Article Title: Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein
Figure Lengend Snippet: Daidzein changes DNA methylation at the BRF2 promoter specifically. CpG methylation- sensitive restriction enzymes cut sites are shown. Transcription start site (TSS) is indicated by +1. MethPrimer program was used to predict the location of CpG islands within the BRF2 (402–556 bp) and BRF1 (50–910 bp) promoters, as indicated by black arrow. Black bars denote the binding sites for primers used in the methylation profile analysis. a-d MCF-7 and ( e ) MDA-MB-231 cells were treated with 0, 3, 10 μM daidzein for 48 h. The genomic DNA was harvested and digested with methylation-sensitive restriction enzymes with cut sites within the promoter, noted on BRF2 and BRF1 promoter schematic, and as a negative control one methylation sensitive enzyme with no recognition sites in the promoter was used. BspEI and BsRFI do not have recognition sites in BRF2 and BRF1 promoters, respectively. The digestion profile was then analyzed by qPCR using primers spanning the BRF2 ( a , e, f ) and BRF1 ( b ) promoter regions, U6 ( c ) and tRNA i Met ( d ) genes. DNA methylation levels were calculated using ΔΔCt method with RPS13 expression levels used as a reference for normalization. Data presented are average of three independent experiments. Statistical analysis was performed using one-way ANOVA a Tukey’s post-test with a 95 % confidence interval (Graphpad Prism 3.03); * = p
Article Snippet: Restriction enzymes AciI, AscI, BanI, BfuAI, BsrFI, BsrBI, BseYI, BfuAI, BspEI, Cac81, FspI, NciI, NruI (New England Biolabs) and HpyCH4III/Taal (Fermentas) were used.
Techniques: DNA Methylation Assay, CpG Methylation Assay, Binding Assay, Methylation, Multiple Displacement Amplification, Negative Control, Real-time Polymerase Chain Reaction, Expressing