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Quantitation of GAD67 Gene Expression in Prefrontal Cortex of Schizophrenia Patients Using the iCycler iQ Detection System and Molecular Beacons, Rev A

quences were determined by Oligo Analyzer 2.5 software (IDT Corporation). Because the sensitivity and reliability of real-time PCR quantitation are dependent on the specificity of PCR, several different primer pairs were initially designed for GAD67 mRNA detection and tested for specificity. PCR was performed with each of these primer pairs using cDNA derived from a pooled sample of human cortical specimens from 10 randomly selected cases (see below for cycling conditions). The PCR products were analyzed on ethidium bromide-stained agarose gels. The primer pair that generated the highest yield of specific product without any nonspecific bands was chosen for the real-time PCR experiments. A similar approach was undertaken to select primers for β-actin detection.

The molecular beacons were designed according to the guidelines available at http://www.molecular-beacons.org. They consisted of probe sequences 2425 nucleotides long, 6-nucleotide arm sequences, the fluorophore FAM covalently linked to the 5' end, and the quencher DABCYL linked to the 3' end. The melting temperatures of the hairpin stems and probe sequences were 6366C. To estimate the stability of the molecular beacons hairpin stem structure, the DNA folding program m-fold (available at http://www.bioinfo.math.rpi.edu/~mfold/dna/form1.cgi) was utilized. Selected primer and probe sequences for this study are shown in Table 2.

Real-Time PCR
Real-time PCR analysis was performed using the iCycler iQ detection system. The conditions of the reaction were carefully optimized by varying molecular beacon (50250 nM), primer (2001,000 nM), and Mg2+ (35 mM) concentrations and the annealing temperature (5560C). The optim
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