Identification of endogenous reference genes for the analysis of microRNA expression in the hippocampus of the pilocarpine-induced model of mesial temporal lobe epilepsy

Autor: Daniel Leite Góes Gitaí, Norberto Garcia-Cairasco, Jamile Taniele-Silva, Fernanda Maria de Araújo Souza, Tiago Gomes de Andrade, Thalita Ewellyn Batista Sales Marques, Maria Luísa Paço-Larson, Mykaella Andrade de Araújo
Jazyk: angličtina
Rok vydání: 2014
Předmět:
Male
Hippocampus
Gene Expression
lcsh:Medicine
Bioinformatics
Epileptogenesis
Biochemistry
Status Epilepticus
Reference genes
Nucleic Acids
Temporal Lobe Epilepsy
Gene expression
Molecular Cell Biology
Medicine and Health Sciences
lcsh:Science
Mammals
Multidisciplinary
Pilocarpine
Animal Models
Reference Standards
Neurology
Vertebrates
Research Article
Normalization (statistics)
EPILEPSIA DO LOBO TEMPORAL
Computational biology
Biology
Research and Analysis Methods
Real-Time Polymerase Chain Reaction
Rodents
Model Organisms
microRNA
Genetics
Animals
Rats
Wistar
Gene
Epilepsy
Gene Expression Profiling
lcsh:R
Organisms
Biology and Life Sciences
Reproducibility of Results
Cell Biology
Rats
Gene expression profiling
Disease Models
Animal
MicroRNAs
Epilepsy
Temporal Lobe
RNA
lcsh:Q
Molecular Neuroscience
Neuroscience
Zdroj: PLoS ONE, Vol 9, Iss 6, p e100529 (2014)
PLoS ONE
Repositório Institucional da USP (Biblioteca Digital da Produção Intelectual)
Universidade de São Paulo (USP)
instacron:USP
ISSN: 1932-6203
Popis: Real-time quantitative RT-PCR (qPCR) is one of the most powerful techniques for analyzing miRNA expression because of its sensitivity and specificity. However, in this type of analysis, a suitable normalizer is required to ensure that gene expression is unaffected by the experimental condition. To the best of our knowledge, there are no reported studies that performed a detailed identification and validation of suitable reference genes for miRNA qPCR during the epileptogenic process. Here, using a pilocarpine (PILO) model of mesial temporal lobe epilepsy (MTLE), we investigated five potential reference genes, performing a stability expression analysis using geNorm and NormFinder softwares. As a validation strategy, we used each one of the candidate reference genes to measure PILO-induced changes in microRNA-146a levels, a gene whose expression pattern variation in the PILO injected model is known. Our results indicated U6SnRNA and SnoRNA as the most stable candidate reference genes. By geNorm analysis, the normalization factor should preferably contain at least two of the best candidate reference genes (snoRNA and U6SnRNA). In fact, when normalized using the best combination of reference genes, microRNA-146a transcripts were found to be significantly increased in chronic stage, which is consistent with the pattern reported in different models. Conversely, when reference genes were individually employed for normalization, we failed to detect up-regulation of the microRNA-146a gene in the hippocampus of epileptic rats. The data presented here support that the combination of snoRNA and U6SnRNA was the minimum necessary for an accurate normalization of gene expression at the different stages of epileptogenesis that we tested.
Databáze: OpenAIRE
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