The transcript level in each sample should be identical for allele 1 and allele 2 unless there is a cis-regulatory effect of one allele that changes its relative expression level. Therefore, one allele serves as the control for the other. To evaluate the amplification efficiency for allelic mRNA expression, we first set up a dilution series using one sample (cDNA or gDNA) that was homozygous for allele 1 and another sample that was homozygous for allele 2. The RT-PCR efficiency for each allele ranges from 95% to 100% with the cDNA sample. In the gDNA sample, the amplification efficiency ranges from 94% to 100%. Real-time data were analyzed using the comparative Ct method [28] and were corrected for the PCR efficiency of each assay. In each cDNA sample, the delta Ct (Ct value of allele 1 - Ct value of allele 2) value was calculated as an average of triplicate reactions. In gDNA samples, delta Ct values were calculated as an average of duplicate reactions. Only the samples with ≤10% standard errors in dCt were used for further analysis.
