in serial dilutions of cDNA derived from a pooled sample (see above) was measured (Figure 2A). Figure 2B shows C_T) values of these amplifications plotted against the logarithm of the relative initial amount of the cDNA. Theoretically, there should be an inverse linear relationship between the C_T) and the logarithm of the number of target molecules that were present initially; the threshold cycles of each replicate group should be separated by exactly 3.32 cycles (antilog210). In the experimental data shown in Figure 2, a linear relationship between C_T) and the initial amount of GAD67 mRNA was demonstrated for 3 orders of magnitude. The mean separation between the groups (the slope of the curve) was -3.03, which is very close to the theoretical value. These data demonstrate the broad dynamic range of GAD67 mRNA quantitation. Similar results were obtained for the β-actin mRNA amplification assay.

Quantitation of GAD67 Gene Expression
The PCRs were quantitated by selecting the amplification cycle where the PCR product of interest was first detected (C_T)). To account for different degrees of RNA degradation and other technical artifacts, the relative quantitation of GAD67 mRNA expression was performed as described in User Manual #2 (comparative C_T) method in separate tubes; Applied Biosystems). The expression level of GAD67 was normalized to the expression level of the endogenous reference (β-actin) in each sample. This relative value was further normalized to the relative expression of GAD67 in the pooled sample. Pooled cDNAs were run in every plate simultaneously with experimental samples. To avoid competition, GAD67 and
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