Primers were based upon previously published primer sets [24-26] or designed using MacVector, which were designed from either mRNA or cDNA to avoid the amplification of potentially contaminating genomic DNA in the total RNA sample (Table 1). Samples of total RNA (0.5 μg) from pooled mouse livers (n = 4) were reverse transcribed according to the SuperScript™ First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) using random hexamers with a final total reaction volume of 20 μl. Quantitative RT-PCR was performed on a PE Biosystems GeneAmp® 5700 sequence Detection System (Forest City, CA, USA). Standard reaction volume was 10 μl containing 1 × QuantiTect SYBR Green PCR master mix (Qiagen, Valencia, CA, USA), 0.002 U AmpErase UNG enzyme (PE Biosystems, Forest City, CA, USA), 0.7 μl of cDNA template from reverse transcription reaction as above and 100–500 nM of oligonucleotide primers. Initial steps of RT-PCR were two minutes at 50°C for UNG activation, followed by a 15 minute hold at 95°C. Cycles (n = 40) consisted of a 15 second melt at 95°C, followed by a 1 minute annealing/extension at 60°C. The final step was a 60°C incubation for 1 minute. At the end of the run, samples were heated to 95°C with a ramp time of 20 minutes to construct dissociation curves to ensure that single PCR products were obtained. All reactions were performed in triplicate. Threshold cycle (CT) analysis of all samples was set at 0.5 relative fluorescence units. The relative quantities of message of genes of interest from the mouse liver samples used in real-time RT-PCR were normalized to cyclophilin to compensate for variations in input RNA amounts. The data were analyzed using the comparative threshold cycle method (CT). Briefly, the CT values are averaged, from the triplicates defining a Δ-CT value calculated by taking the average CT of the gene of interest and subtracting it from the average CT of cyclophilin. The ΔΔ-CT was calculated by subtracting the average Δ-CT(calibrator) values from the Δ-CT(sample). The relative quantification was then calculated by the expression 2-AverageΔΔ-CT. The mRNA quantity for the calibrator (wild type) was expressed as 1 and all other quantities were expressed as a fold difference relative to the calibrator (wild type).