Habano et al. BMC Cancer 2011, 11:81 http://www.biomedcentral.com/1471-2407/11/81 Page 3 of 10 Annealing temp.(°C) 55 55* 55 55 55* 55 55 60 60 55* Product size (bp) 574 602 230 318 209 292 438 400 373 273 Figure 1 Location of the CpG sequences examined. CpG sites (vertical bars), CpG islands identified by CpG island searcher (horizontal thick lines) and transcription start sites within the 5’ prime region (curved arrows) are shown in the (a), PXR; (b), CYP3A4; (c), VDR; and (d), PRMT1 genes. The segments indicated by double-pointed arrows were examined for DNA methylation by bisulfite direct sequencing. Table 1 Primers used for DNA methylation analysis Gene Segment Primer sequence (5’-3’) PXR promoter PXR exon 3 CYP3A4 5’UTR VDR promoter PRMT1 promoter 1F 1R 2F 2R 3F 3R 4F 4R COBRA F R F R 1F 1R 2F 2R F R GAAGATAATTGTGGTTATTTTTTGGTA CCACCTCCCTAAATAATATTACT GTTGTTTTTAGTGGTAAAGGATAGA CACACACATCTTTTAACTAAAACT AGGATTTATTATTTTAAGGAGGGGTT CCTTAAAACAATACCTCTAACCAT GTAAGATTTGGAGATTTTTTATATTTG CTATCCTTCTCTACTAATAAAAATAC GTAGGGAGAATATAATGAGAATAA ACTAAAATAAAAACAATACTTCCTCTTCAC ATTTTTTTATAGGAGGGTTATGAAA TACACACRAACACCAACTCACATAT GAGTTATGGTGGGTTTTATTTAG TCTACATTTCCATCTAAAATACC ATAATTTTAGGTTTTAGGAGGTAG CCTAAACTAACCAAACCAAAACTT GGGTTGTTTTTGTTTGTTAAAAGG CTTATTACCCAAATACTAAACACT AGGAGAAAGGGGGGGTTTTGGT AACCCTTAAAAACTAAAAAACC F, forward primer; R, reverse primer. *Dimethyl sulfoxide (5%) was added to the PCR mixture.