The spinal cord samples were dissected and stored as described above. For RNA and real-time quantitative PCR, total RNA was isolated from spinal cord using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The purity and concentration were determined spectrophotometrically. Reverse transcription was accomplished using a First Strand complementary DNA Synthesis Kit (Invitrogen, Carlsbad, CA). Real-time PCR was performed in an ABI prism 7900HT system (Applied Biosystems, Foster City, CA). All PCR experiments were performed using the SYBR Green I master kit (Applied Biosystems). The Dcc primers were purchased from SABiosciences (Catalog number: PPM03679F, SABiosciences, Valencia, CA). The sequences for the GAPDH primers were (forward primer) 5-CTGGAGAAACCTGCCAAGTATGATG-3 and (reverse primer) 5-GAGACAACCTGGTCCTCAGTGTAGC-3. Quantification was accomplished according to the standard curve method. In order to achieve the same PCR efficiency for each analyte, 1:10 serial dilutions of cDNA were used to construct standard curves for Dcc and GAPDH. The R2 values for the standard curves for each analyte approached 1.0, suggesting the same amplification efficiency in the PCR reactions under these conditions. Melting curves were performed to document
