Abstract
We used reverse transcription-coupled PCR to produce a high-resolution temporal map of fluctuations in mRNA expression of 112 genes during rat central nervous system development, focusing on the cervical spinal cord. We have used an established RT-PCR protocol (4) to measure the expression of 112 genes in CNS development. Cervical spinal cord tissue was dissected from triplicate animals or litters (Sprague-Dawley albino rats), in accordance with National Institutes of Health guidelines, from embryonic days 11 through 21 (E11-E21; determined by crown-rump length), postnatal days 0-14 (P0-P14), and adult (P90 or adult). Gene-specific primers were designed from GenBank sequences by using the OLIGO software (National Biosciences, Plymouth, MN). RNA isolated from tissue samples by using RNAstat 60 (Tel-Test, Friendswood, TX) was adjusted to 200 ng/l according to absorption at 260 nm, before RT-PCR (Perkin-Elmer GeneAmp RNA PCR kit, Applied Biosystems); PCR involved preheating a mixture of Taq antibody (TaqStart, CLONTECH), primers, cDNA, and PCR components to 97C for 90 sec before amplification.The PCR cycle was 30 sec at 95C (dissociation), 45 sec at 60C (annealing), and 60 sec at 72C (extension). Amplification was within the exponential range (4). [J Physiol Sci. 2007;57 Suppl:S144]
