EXPRESSION PROFILING OF MESSENGER-RNA OBTAINED FROM SINGLE IDENTIFIEDCRUSTACEAN MOTOR-NEURONS - DETERMINATION OF SPECIFICITY OF HYBRIDIZATION

The purpose of this study was to determine if the technique of express ion profiling would allow us to determine the changes in the abundance of certain mRNAs in identifiable single neurons as a result of height ened electrical activity. In doing so we developed an approach to test the specificity of hybridization in expression profiling. Messenger R NA from single identified crayfish motor neurons was amplified by liga tion-mediated reverse transcription PCR and hybridized by dot-blotting to 45 target cDNAs from different species. As a test of specificity, the hybridization was repeated using unlabelled cDNAs, the dots were e xcised, and the hybridized nucleic acids were re-amplified, cloned, an d sequenced to confirm their identity. By cloning and sequencing the r e-amplified product for each cDNA examined, one can determine the degr ee of background hybridization as compared to homologous hybridization . False positive results were also observed when a species-specific cD NA and highly stringent hybridization conditions were used. Our result s demonstrate that ligation-mediated PCR is a useful technique for che cking the specificity of expression profiling. This approach can easil y be adapted to any situation when confirmation of the specificity of nucleic acid hybridization is required. During this study, pare of a n ovel crayfish neuronal actin cDNA was cloned and sequenced.