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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

in-shaped molecules with a terminally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid (Figure 1). They are designed in such a way that the loop portion is a probe sequence complementary to a target DNA molecule. The stem is formed by the annealing of complementary arm sequences on the ends of the probe molecule. A fluorescent moiety is attached to the end of one arm and a quenching moiety is attached to the end of the other arm. The stem-loop structure keeps these 2 moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer.

At the annealing temperature of the probe, it forms a hybrid with the target molecule that is longer and more stable than the stem, causing the fluorophore and the quencher to move away from each other and leading to increased fluorescence. This can be measured by the iCycler iQ detection system. Molecular beacons that do not bind to the target return to the stem-loop structure and are dark. Detailed directions on molecular beacon synthesis and design are available at http://www.molecular-beacons.org.


Methods
Human Postmortem Tissue
Frozen postmortem brain samples of subjects diagnosed antemortem with schizophrenia (n = 26) by DSM-IV criteria, and of normal elderly controls (n = 13), were obtained from the Mount Sinai/Bronx Veterans Administration Medical Center Department of Psychiatry Brain Bank. The mean age, PMI, tissue pH, and sex distribution of the cohorts used in this study are shown in Table 1. Gray matter from the frozen dorsolateral prefrontal cortex, Brodmann area 46 (Rajkowska and Goldman-Rakic 1995), was dissected
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