Comparison of the sizes of cloned and sequenced AP-PCR products to the sizes of unique RAMs. After the sequences were obtained, the sizes of the cloned products were compared with the sizes of the unique RAMs in order to determine which cloned products represented unique precancerous for and tumor RAMs. In many instances, it can be confidently stated that a particular cloned product represents a single RAM and as such, the methylation status of that RAM is unambiguous. However, the raw data analysis performed to establish if a RAM occurred in a treatment group as compared with its respective control group (Phillips et al., 2007) is based upon the understanding that the ABI 3130 Genetic Analyzer capillary electrophoresis instrument does not detect PCR product sizes with 100% accuracy.

Therefore, in certain instances during the analysis of the raw data, PCR product sizes were combined. Six animals per experimental group were used and restriction digestions were performed in duplicate, followed by AP-PCR, for a total of 12 reactions. If multiple PCR product sizes within two base pairs of one another displayed product in less than half of the 12 AP-PCR reactions, these products were considered to be ��identical�� and were subsequently combined. This procedure has implications for analysis of the cloning data. For example, two RAMs occurred uniquely in the tumor tissue as a result of RsaI/HpaII digestion: a hypomethylation at 402 bp and a carry forward new methylation at 404 bp (Supplemental Fig. S1). A carry forward methylation change is a unique RAM that was observed in both precancerous and tumor tissue.

A PCR product of 404 bp was cloned, and a BLAT search showed that the product spans an intronic region within the transmembrane protein 132d (Tmem132d) gene (Table 1). Due to our basic data analysis �� 2 bp assumption, the methylation status of Tmem132d is ambiguous; it could represent the hypomethylated RAM at 402 bp or the newly methylated carry forward RAM at 404 bp. TABLE 1 Genes and Genomic Regions Identified from Unique PB-induced RAMs in C3H/He CAR WT (Precancerous Liver and/or Liver Tumor), as compared with Resistant PB-Treated KO Mice, were Cloned and Subjected to BLAST-like Alignment Tool (BLAT) Searches Additionally, Batimastat the use of three different methylation-sensitive enzyme pairs could also reveal multiple methylation statuses of a particular gene. In the case of dipeptidylpeptidase 10 (Dpp10), RsaI/HpaII digestion revealed a hypomethylated RAM, whereas RsaI/MspI digestion demonstrated a carry forward hypermethylated RAM in the precancerous tissue (Table 1).