RESUMEN
Low voltage-activated calcium channels are thought to play a key role in the generation of spike and waves discharges characteristic of absence epilepsy. Therefore, the expression level of mRNA encoding calcium channel alpha1E and alpha1G subunits was measured in the brain of genetic absence epilepsy rats from Strasbourg (GAERS). Using quantitative RT-PCR and in situ hybridization, no difference was found in alpha1G mRNA expression between GAERS and control animals, while a decreased expression of alpha1E was seen in the cerebellum and the brain stem of the GAERS. This phenomenon was not observed in young animals when the epileptic phenotype is not expressed.
Asunto(s)
Canales de Calcio/genética , Epilepsia Tipo Ausencia/metabolismo , Proteínas del Tejido Nervioso/genética , ARN Mensajero/metabolismo , Animales , Tronco Encefálico/metabolismo , Canales de Calcio Tipo R , Proteínas de Transporte de Catión , Cerebelo/metabolismo , Epilepsia Tipo Ausencia/genética , Hibridación in Situ , Isomerismo , Masculino , Ratas , Ratas Endogámicas/genética , Ratas Wistar , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Quantitative studies are commonly realised in the biomedical research to compare RNA expression in different experimental or clinical conditions. These quantifications are performed through their comparison to the expression of the housekeeping gene transcripts like glyceraldehyde-3-phosphate dehydrogenase (G3PDH), albumin, actins, tubulins, cyclophilin, hypoxantine phsophoribosyltransferase (HRPT), L32. 28S, and 18S rRNAs are also used as internal standards. In this paper, it is recalled that the commonly used internal standards can quantitatively vary in response to various factors. Possible variations are illustrated using three experimental examples. Preferred types of internal standards are then proposed for each of these samples and thereafter the general procedure concerning the choice of an internal standard and the way to manage its uses are discussed.
Asunto(s)
Expresión Génica , Genes/genética , ARN Mensajero/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 28S/genética , Animales , Biotecnología/normas , Citocinas/biosíntesis , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Ratones , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Proteínas Ribosómicas/biosíntesisRESUMEN
Differential mRNA display was carried out to find genes that are differentially regulated in the brain of a rat strain with absence epilepsy, the genetic absence epilepsy rats from Strasbourg (GAERS). Among the 32 differentially displayed cDNA fragments actually cloned and sequenced, one shows 100% identity with the rat heavy chain ferritin (H-ferritin) mRNA. Northern blot analysis confirmed the up-regulation of the H-ferritin mRNA. Using dot blotting, a 40% increase in expression was reported in the subcortical forebrain of the adult GAERS, while cortex, brain stem, and cerebellum appeared unmodified. This change was not observed in the brain of 25-day-old rats, an age at which the epileptic phenotype is not present. By in situ hybridization, the enhanced expression was localized in the hippocampus. The increase in mRNA encoding H-ferritin was not immunodetected at the protein level by Western blotting. These results are not apparently related to the neural substrate of SWD or to the distribution of local increase in glucose metabolism previously described in the GAERS. It is hypothesized that the up-regulation of the H-ferritin mRNA is part of a mechanism protecting the hippocampus, a seizure-prone area, against a possible overactivation during absence seizures.