,

2004), respectively. Genes were annotated with transcription start-site (TSS) using REFLINK and REFFLAT tables downloaded from the UCSC genome browser data on August 23, 2010 (Karolchik et al., 2003). Within each AHRE-motif, a PhyloHMM conservation score was calculated across different species. The scores vary from 0 to 1, with a score of 0 meaning NVP-BGJ398 that there is a minimal conservation and 1 meaning that there is a strong conservation. Motifs that are evolutionarily well conserved are particularly likely to be functional (Siepel and Haussler, 2004). Primers and probes were designed using the real-time PCR Assay Design Tool on the Integrated DNA Technologies (IDT, Coralville, IA) website (http://www.idtdna.com/Scitools/Applications/RealTimePCR). To ensure specificity, probes were compared with Rattus norvegicus nr/nt database using nucleotide Basic Local Alignment Search Tool (BLAST). Similarly, primer pairs were compared to the same database using Primer BLAST (http://www.ncbi.nlm.nih.gov/blast) ( Altschul PFI-2 et al., 1990 and Altschul et al., 1997). Total RNA samples were reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied

Biosystems). In addition, for each reverse-transcribed sample, a similar preparation was made where all the reagents were included except for the reverse transcriptase.

The above reactions were conducted with 1 μg of RNA as outlined in the manufacturer’s instruction. PCR reactions were prepared with 5 ng of cDNA using the TaqMan Gene Expression Master Mix (Applied Biosystems) with gene-specific primers/probes ( Table 1). A total of 84 biological replicates were analyzed for H/W rats and 68 replicates for L-E rats, each performed Methane monooxygenase with two technical replicates in a 10 μL reaction volume. PCR reactions were run on the 7900HT Fast Real-Time PCR System (Applied Biosystems) using default settings for relative quantification calculated from comparative Ct values. qPCR results were then collected using Sequence Detection System Software v2.3 (Applied Biosystems) and the quantitated data were loaded into the R statistical environment (v2.12.2). These data were then processed and normalized as previously described ( Barsyte-Lovejoy et al., 2006) using the previously validated housekeeping genes Gapdh and Pgk1 ( Pohjanvirta et al., 2006). Normalized data were log2-transformed and visualized across the different doses and time points tested for both L-E and H/W rats.