, 2004). Protocol Employed for the Annotation of PB-Induced Unique Rams in Precancerous and Tumor Tissue The RAMs cloned and annotated in the current study were previously detected via an approach involving table 5 methylation-sensitive restriction digestion, arbitrarily primed PCR (AP-PCR), and capillary electrophoresis (CE) (Supplemental Fig. S1, originally from Phillips et al., 2007), a technique described in detail by Bachman et al. (2006a). The following comparisons were previously made between experimental groups: (1) the CAR KO, 23-week PB data were compared with the CAR KO, 23-week control data, and (2) both the CAR WT, 23-week PB (precancerous tissue) and CAR WT, 32-week PB (tumor tissue) data were compared with the CAR WT, 23-week control data.

For each specific PCR product size that was observed in control versus PB-treated an individual Student’s t-test was performed to evaluate whether or not there was a statistical (p < 0.05) difference in the peak area. Additionally, new methylations (PCR products which were observed in the PB-treated and not in control), as well as 100% hypomethylations (PCR products which were observed in control but not seen at all in PB treated), were viewed as being ��significant�� (Bachman et al., 2006a). For the methylation analysis performed by Phillips et al. (2007), whole liver from four of the five (not including the WT, 32-week PB) groups was utilized; the precancerous liver tissue (WT, 23-week PB) contained no tumors, however, based upon histology of adjacent tissue, there are expected to be very numerous microscopic foci of cellular alteration diffused throughout the tissue.

Importantly, DNA was isolated from individual liver tumors that developed in the WT, 32-week PB group. The 23-week PB-treated mice are 30 weeks of age (PB treatment started when the animals were 7 weeks old), and the 32-week mice are 39 weeks of age. Thus, the mice were past the juvenile development stage and not into old age, and at an age where a reasonable degree of stability of methylation might be anticipated over a 9-week period. Cloning and sequencing of AP-PCR products. AP-PCR products were first cloned using an in-gel approach to identify in what regions of the genome the PB-induced unique precancerous and tumor RAMs occurred. The AP-PCR products and a 100 base pair DNA ladder (Invitrogen, Carlsbad, CA) were electrophoresed through a 3% NuSieve GTG low melting temperature agarose gel (Lonza Biosciences, Basel, Switzerland).

Portions of the gel that contained PCR products within 100 base pair size ranges were excised, melted and used Batimastat for in-gel cloning reactions prepared with the pGEM-T Easy Vector Kit (Promega, Madison, WI). Clones that contained PCR product inserts were purified and sequenced at the Research Technology and Support Facility at Michigan State University.