Detection of gene expression in single neurons by patch-clamp and single-cell reverse transcriptase polymerase chain reaction.
Detection and quantitation of gene expression in single cells is especially important in the central nervous system where, at the cellular level, the synapse can be considered the single functional unit. For example, the consolidation of long-term memories may be mediated by persistent changes in the strength of synaptic transmission at individual synapses. In order to investigate the requirement for de novo RNA synthesis during long-term potentiation in individual neurons, we have combined single-cell electrophysiology with single-cell gene-expression methodology. Described are methods combining whole-cell patch-clamp and single-cell RT-PCR for the detection of a single mRNA species for nitric oxide synthase, or, through a multiplex strategy, for the simultaneous detection of several mRNAs including heme oxygenase 2, protein phosphatase inhibitor 1 protein, and several isoforms of the calcium/calmodulin dependent protein kinase II.
Chiang
L W
LW
Department of Neurobiology, Stanford University Medical Center, CA 94305-5401, USA. chiang@mpi.com
D000818:Animals; D015870:Gene Expression; D006624:Hippocampus / Q000166:cytology; D009474:Neurons / Q000378:metabolism* / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000235:genetics; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012194:RNA-Directed DNA Polymerase; D015398:Signal Transduction; D009435:Synaptic Transmission
D012333:RNA, Messenger; D019001:Nitric Oxide Synthase; D012194:RNA-Directed DNA Polymerase