Because three different restriction digestions were utilized, multiple selleckbio RAMs might represent the same gene/uncharacterized region. The following criteria needed to be met in order for a gene/uncharacterized genomic region identified from a PB-induced RAM to be viewed as being in common between the studies: (1) the RAM must have been cloned in both the B6C3F1 (2 and/or 4 weeks PB) and CAR WT (precancerous and/or tumor) mice, and (2) the unique RAMs in the B6C3F1 and CAR WT mice must have aligned to the same region of the genome. Hypermethylated RAMs (significant increases, Student’s t-test, p < 0.05) and newly methylated RAMs are considered to be increases in methylation, whereas hypomethylated RAMs (both 100% decreases, and those which are significant, Student's t-test, p < 0.

05) are considered to be decreases. Genes and uncharacterized genomic regions, identified from identical, unique PB-induced RAMs that formed in both CAR WT (precancerous liver and/or liver tumor) and B6C3F1 (2 and/or 4 weeks treated), are listed in Table 3 and Supplemental Table S3, respectively. TABLE 3 Genes Identified from Identical, Unique PB-induced RAMs that Formed in both CAR WT (Precancerous Liver and/or Liver Tumor) and B6C3F1 (2- and/or 4-Week Treated) Mice. Cloning and sequencing revealed two observations regarding criterion number 2 (above). First, following digestion with the same methylation-sensitive restriction enzyme, PCR products occasionally formed which (1) represented distinct RAMs that differed by more than 2 bp in multiple groups (B6C3F1, 2 and/or 4 weeks, and CAR WT, precancerous liver and/or liver tumor) and (2) aligned to the same region of the genome.

Second, following digestion with the same methylation-sensitive restriction enzyme, PCR products occasionally formed which (1) represented distinct RAMs that were within GSK-3 2 bp of one another (e.g., unique RAMs at 315 and 317 bp) in multiple groups (B6C3F1, 2 and/or 4 weeks, and CAR WT, precancerous liver and/or liver tumor) and (2) aligned to the same region of the genome. Thus, for a particular restriction digestion, unique RAMs in the B6C3F1 and CAR WT mice that were within 2 bp of one another or more than 2 bp apart, which were represented by PCR products that aligned to the same region of the genome, were considered to be one RAM. This is evident in Table 3 and Supplemental Table S3. The following is an example of situation number 1, above. A PCR product, representing a 464-bp RAM that was identified after RsaI/MspI digestion and AP-PCR, was cloned in the B6C3F1 mice at 4 weeks, and aligned to Bcat2 (Table 3).