The copy number of CYP2E1 was determined for each sample in quadruplicate through the amplification of both a probe specific to CYP2E1 and a standard probe by real-time PCR using the standard Gene Dosage protocol provided by Applied Biosystems (Livak and Schmittgen, 2001). Preliminary amplification showed the two probes used for analysis had different efficiencies of amplification, which was corrected by a standard dilution curve added to each plate. The fold increase after n number of cycles was calculated by (efficiency)n and the ratio of this increase between the target and reference genes provided copy number. Standard copy number quantification assumes equal amplification efficiencies, but this is not always a valid assumption. Correcting for even small differences in amplification efficiencies leads to less variability among the quadruplicate samples and lowered standard error in overall copy number determination.
