Research Report The combined use of non-radioactive in situ hybridization and real-time RT-PCR to assess gene expression in cryosections Corinna Haupt a , Else A. Tolner b,1 , Uwe Heinemann b , Otto W. Witte a , Christiane Frahm a, ⁎ a Department of Neurology, Friedrich-Schiller-University, Jena, Germany b Institute of Physiology, Charité, Berlin, Germany ARTICLE INFO ABSTRACT Article history: Accepted 9 August 2006 Available online 20 September 2006 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Computerized image analysis Connexin 43 Kainic acid Seizure Status epilepticus Abbreviations: CP, crossing point Cx43, connexin 43 DIG, digoxigenin E, PCR efficiency GAPDH, glyceraldehyde 3-phosphate dehydrogenase HC, hippocampus PCR, polymerase chain reaction PFA, paraformaldehyde R, relative expression ratio SDS, sodium dodecyl sulfate SE, status epilepticus SSC, saline sodium citrate TEA, triethanolamine BRAIN RESEARCH 1118 (2006) 232 – 238 ⁎ Corresponding author. Fax: +49 3641 9 325902. E-mail address: christiane.frahm@med.uni-jena.de (C. Frahm). 1 Present address: Dept. of Biological and Environmental Sciences, University of Helsinki, Finland. 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.08.037 available at www.sciencedirect.com www.elsevier.com/locate/brainres