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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

levels in cells, whereas GAD65 can be rapidly activated in times of high GABA demand.

The goal of our study was to compare GAD67 gene expression in dorsolateral prefrontal cortex of postmortem specimens from chronically ill elderly schizophrenia patients to normal controls matched for age and postmortem interval (PMI). Real-time PCR* was applied to quantitate the expression of the gene. Real-time PCR necessitates the use of fluorogenic probes. Reactions are quantitated by the point in time during cycling when amplification of PCR product is first detected (the threshold cycle, or CT) rather than by the amount of PCR product accumulated after a fixed number of cycles. The early cycles of PCR are characterized by an exponential increase in target amplification. As reaction components become limiting, the rate of target amplification decreases until a plateau is reached and there is little or no net increase in the PCR product. The iCycler iQ detection system allows for product formation to be visualized by monitoring fluorescent reporter molecules in the PCR. In this way the exponential phase of the amplification can be determined. The point at which fluorescence significantly increases above background is described as the threshold cycle (CT)). At the CT), the PCR amplification is still in the exponential phase and none of the reaction components are limiting. CT) values are very reproducible for reactions with the same starting copy number. This leads to greatly improved precision in the quantitation of DNA.

Molecular beacons were used as fluorogenic probes in our real-time PCR experiments. Molecular beacons (Marras et al. 1999, Tyagi and Kramer 1996) are hairp
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