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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

al 25 l PCR contained 5 l of the relevant cDNA (diluted 25-fold with water), 200 nM of molecular beacon for GAD67 or 100 nM for β-actin, 500 nM of forward and reverse primers, 1 U of AmpliTaq Gold DNA polymerase (Applied Biosystems), 250 M of each dNTP, 4 mM MgCl2, 50 mM KCl, and 10 mM Tris-HCl (pH 8.3). The thermal cycling program consisted of 10 min at 95C to activate the polymerase, followed by 10 cycles of 15 sec at 95C, 30 sec at 6859C (touchdown PCR, annealing temperature was decreased 1C after each cycle), and 30 sec at 72C. This touchdown step was followed by 35 cycles of 15 sec at 95C, 30 sec at 58C, and 30 sec at 72C. Fluorescence was monitored during the 58C annealing steps.

Assessment of DNA Contamination in RNA Samples
In order to measure the level of contaminating chromosomal DNA, all RNA samples not treated with reverse transcriptase and their respective cDNA samples were subjected to PCR using GAD67 primers. The products of the PCR reactions were analyzed on ethidium bromide-stained agarose gels. In contrast to their respective cDNA templates, no PCR products could be detected with gel electrophoresis when RNA samples were used. Random RNA samples (n = 10) were also subjected to real-time PCR using β-actin primers and a molecular beacon. The RNA samples showed at least 3 orders of magnitude lower amounts of the initial template molecules than their respective cDNAs (the difference in CT) between the RNA and cDNA in each sample was at least 10 cycles), demonstrating negligible amounts of genomic DNA contamination (not shown).

Dynamic Range of Real-Time PCR
To determine sensitivity of the assays, the amplification of each mRNA
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