( r accumulation under fluorescent micros...)Laser Capture Microdissection (LCM) and Expression Profiling of Long-Range Projection Neurons

G. Taqman quantitative PCR to validate differential gene expression on the microarrays
NOTE: Taqman Quantitative PCR was performed with normalized inputs of amplified RNA, based on measurements from the NanoDrop ND-1000 spectrophotometer. It was assumed that the reverse transcription step went to completion.
1. Based on the sequence information for the clones identified as differentially expressed on the microarrays, primers and probes were designed using Primer-Express software (Applied Biosystems). Primers and probes were then ordered with these sequences.

2. Primers and probes were diluted according to manufacturers instructions (Applied Biosystems) and combined with the appropriate volume of Taqman master mix. Highly reproducible results were obtained using 900 nM of each primer and 250 nM of probe.

3. 22.5 L of the master mix was loaded for each gene of interest on a 96-well optical reaction plate, and 2.5 L of cDNA template from each projection neuron sample was added appropriately. The plate was placed into the Applied Biosystems 7900HT Fast Real-Time PCR System and the PCR was run using the following parameters: 95˚C denaturation, 60˚C annealing/extension for 40 cycles.

4. To generate the standard curve, 2.5 L of serially diluted standard was added to 22.5 L of master mix. Copy numbers of aRNA for the sequence of interest were determined by creating standard curves with purified, single-stranded DNA coding for the sequence of interest. This DNA was serially diluted 10fold five to six times and run in separate q-PCR reactions on the same plate as the unknowns. Copy numbers of the standards were calculated based on the molecular weight of the standard, and a standard curve was generated from the Taqman data by plotting cycle threshold (y-axis) against copy number (x-axis) for the serially diluted stand
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