To directly compare COMT gene expression levels, we optimized a proven two-step, high-temperature, semi-quantitative RTPCR protocol (denature at 94°C for 30 seconds followed by extension at 71°C for 40 seconds for 33 cycles) [12] using primers specific for either the 675G or 675A: the forward 675G primer (COMT-ValF 5′-ATGGTGGATTTCGCTGGCGT-3′) and the 675A primer (COMT-MetF 5′-ATGGTGGATTTCGCTGGCAT-3′) were positioned within exon 4 of the COMT gene, and the reverse primer (COMT-R 5′-CTTCCGCAGCAGGCCACATT-3′) was positioned in exon 5. Only the patient (Figure 2(b), lane 2) expressed the 675A allele and both the patient and two normal controls (lanes 3 and 4) had comparable levels of expression of the 675G allele, as evident from the agarose gel electrophoresis of the amplicons.
