title: The combined use of non-radioactive in situ hybridization and real-time RT-PCR to assess gene expression in cryosections.
abstract: Gene expression changes in pathophysiological states can be spatiotemporally monitored by in situ hybridization and reliably quantified by real-time RT-PCR. Here we developed a new method whereby adjacent slides of frozen sections can be used for gene expression analysis by in situ hybridization and real-time RT-PCR. We applied this method to assess the mRNA expression of connexin 43 (Cx43), the major astrocytic connexin, after kainate-induced seizures in rat hippocampus. Gap junction-building connexins play a role in the pathogenesis of several diseases of the brain, including epilepsy. The number of Cx43 mRNA-positive cells in the hippocampus of kainate-treated and control rats was automatically quantified by computerized image analysis of brain sections hybridized with DIG-labeled RNA probes. In parallel, real-time RT-PCR was used to examine the relative Cx43 mRNA levels in hippocampal tissue from adjacent brain sections. Applying these two very sensitive methods we showed that kainate induced seizures do not affect hippocampal connexin 43 mRNA expression.
authors.affiliation: Department of Neurology, Friedrich-Schiller-University, Jena, Germany.
authors.forename: Corinna
authors.initials: C
authors.lastname: Haupt
authors.forename: Else A
authors.initials: EA
authors.lastname: Tolner
authors.forename: Uwe
authors.initials: U
authors.lastname: Heinemann
authors.forename: Otto W
authors.initials: OW
authors.lastname: Witte
authors.forename: Christiane
authors.initials: C
authors.lastname: Frahm
mesh_terms: D000818:Animals; D002450:Cell Communication / Q000235:genetics; D002452:Cell Count / Q000379:methods; D018031:Connexin 43 / Q000235:genetics; D003292:Convulsants; D004195:Disease Models, Animal; D004827:Epilepsy / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism; D017629:Gap Junctions / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D006624:Hippocampus / Q000033:anatomy & histology / Q000378:metabolism; D019044:Image Cytometry; D017403:In Situ Hybridization / Q000379:methods* / Q000639:trends; D007608:Kainic Acid; D008297:Male; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000639:trends
chemical_list: D018031:Connexin 43; D003292:Convulsants; D012333:RNA, Messenger; D007608:Kainic Acid