Expression profiling of mRNA obtained from single identified crustacean motor neurons: determination of specificity of hybridization.
The purpose of this study was to determine if the technique of expression profiling would allow us to determine the changes in the abundance of certain mRNAs in identifiable, single neurons as a result of heightened electrical activity. In doing so we developed an approach to test the specificity of hybridization in expression profiling. Messenger RNA from single identified crayfish motor neurons was amplified by ligation-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 excised, and the hybridized nucleic acids were re-amplified, cloned, and sequenced to confirm their identity. By cloning and sequencing the re-amplified product for each cDNA examined, one can determine the degree of background hybridization as compared to homologous hybridization. False positive results were also observed when a species-specific cDNA and highly stringent hybridization conditions were used. Our results demonstrate that ligation-mediated PCR is a useful technique for checking the specificity of expression profiling. This approach can easily be adapted to any situation when confirmation of the specificity of nucleic acid hybridization is required. During this study, part of a novel crayfish neuronal actin cDNA was cloned and sequenced.
Pekhletski
R
R
Faculty of Pharmacy, MRC Group in Nerve Cells and Synapses, University of Toronto, Ontario, Canada.
Cooper
R L
RL
Atwood
H L
HL
Hampson
D R
DR
D000818:Animals; D003001:Cloning, Molecular; D003445:Crustacea / Q000235:genetics* / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics; D004594:Electrophysiology; D009046:Motor Neurons / Q000378:metabolism* / Q000502:physiology; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*
D018076:DNA, Complementary; D012333:RNA, Messenger