( equent analyses were performed. ...)Laser Capture Microdissection (LCM) and Expression Profiling of Long-Range Projection Neurons

D. Reverse transcription of the amplified RNA
1. Based on the NanoDrop yield measurements, 2.5 g of aRNA from each type of projection neuron from within the same animal was reverse transcribed in the presence of Cy-dyes using the protocol described in Arcturus Protocol #3. cDNA products were purified as described in Protocol #3 and eluted in 50 L of nuclease-free water. Samples were then concentrated to 12.5 L each. The labeled cDNA was then applied to the microarrays as described in Section E below.

2. For Taqman quantitative PCR analyses, an equivalent amount of aRNA from each type of projection neuron (≤ 0.25 g) and reverse transcribed using the RETROscript Kit per manufacturers instructions. Samples were eluted in 30 L of nuclease-free water. The unlabeled cDNA was diluted 1:10 in water and used in Taqman assays as described in Section G below.

E. Microarray hybridizations, scanning, and analysis
1. 12.5 L of the concentrated, labeled cDNA from each type of projection neuron was applied to the cDNA micro-arrays and allowed to incubate overnight according to the Corning Pronto kit instructions.

2. The hybridized microarrays were scanned at each wavelength for Cy3 and Cy5. Software (e.g. GenePix Pro) was used to obtain the median fluorescent intensity values minus the background for each spot on the microarray.

3. The most differentially expressed genes were identified by either fold-change criteria or statistical analysis using a microarray analysis software package such as GeneTraffic Duo.

F. In situ hybridization to validate differential gene expression found on the microarrays
1. Based on the sequence information for the clones identified as differentially expressed on the microarrays, PCR was used to amplify select clones using oligodT primed cDNA as templat
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