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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

from coronal sections of frozen (-80C) brain. The dissected tissues were pulverized at -190C into a fine powder and aliquotted into individual micro test tubes and stored at -80C until use.

RNA Isolation
Total RNA was isolated from 50 mg of tissue by the guanidinium isothiocyanate method, using a ToTALLY RNA kit (Ambion) according to the manufacturers protocol. To remove genomic DNA contamination, isolated RNA samples were then treated with 40 U DNase I (Ambion) for 1 hr at 37C in a 200 l reaction mixture containing 2.5 mM MgCl2, 0.1 mM CaCl2, 10 mM Tris-HCl (pH 7.5), and 120 U of RNaseOUT enzyme (Invitrogen). RNA samples were then extracted with phenol/chloroform/isoamyl alcohol, precipitated with 100% ethanol, and washed twice with cold 70% ethanol. Total RNA concentration was determined by absorbance at 260 nm. Yield of total RNA ranged from 15 to 30 g per 50 mg of brain tissue. The A260/A280 ratios of the samples were >2.1. The yield and quality of total RNA was also analyzed by agarose gel electrophoresis.

Reverse Transcription (RT) Reaction
Total RNA (~2 g) was used in a 20 l RT reaction to synthesize cDNA using a ThermoScript RT-PCR system kit (LTI-Invitrogen) and random hexamers as primers. RT reactions were performed for 1 hr at 52C, followed by RNase H treatment to remove RNA template, as described in the commercial protocol.

Primer and Molecular Beacon Design
The PCR primers and molecular beacons were synthesized commercially (IDT Corporation, Coralville, IA). PCR primers were designed using Vector NTI software (InforMax, North Bethesda, MD). The melting temperatures (Tm) of the primer and probe se
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