A

11 u

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log starting quantity

Fig. 3. Real-time reverse transcriptase polymerase chain reaction (PCR) of in vitro transcribed choline acetyltransferase messenger RNA (mRNA) using LUX primers. (A) The fluorescence vs PCR cycle for threefold serial dilutions (66-107 copies) of in vitro transcribed mRNA (four replicates per dilution). (B) Standard curve of CT vs initial RNA template for in vitro transcribed mRNA.

treated with DNase I before reverse-transcription to remove any trace of DNA carried over during RNA isolation. Plots of fluorescence vs PCR cycle are generated by the ABI 7700 SDS software (Fig. 4). The cycle threshold (CT) for a fluorescent PCR correlates with amount of initial template in the PCR. The CT values for the PCRs are between 15 and 32 cycles, except the PCRs for GLUR1 and ChAT, which are typically between 28 and 38. The CTs for GAPDH for all time points range between 16.9 and 18.5, which indicates that GAPDH expression is relatively constant. Relative quantification to be performed as a relative fold-increase in transcript level with respect to the time 0 level (pre-induction). This method, called comparative CT or AACT, does not require plotting a standard curve of CT vs starting copy number. Instead, the amount of target is calculated based on the difference (ACT) between the average CT of each time point and the average CT of the 0-time point. Before sub-

10 15 20 25 30 35 40

Fig. 3. Real-time reverse transcriptase polymerase chain reaction (PCR) of in vitro transcribed choline acetyltransferase messenger RNA (mRNA) using LUX primers. (A) The fluorescence vs PCR cycle for threefold serial dilutions (66-107 copies) of in vitro transcribed mRNA (four replicates per dilution). (B) Standard curve of CT vs initial RNA template for in vitro transcribed mRNA.

traction, both CT values are normalized by subtracting the average CT of the endogenous reference gene, GAPDH. The best three of four replicates are used for relative quantitation. The variability among replicates is expressed as the average of the standard deviation for all replicates of each time point of each gene, which is typically in the range of 0.35. The maximum values in standard deviations occur for PCRs with high CTs like amplifying GLUR1 from early time points. See Note 3 for further information on the expression patterns of the genes and a comparison to published results.

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