title: Validation of reference genes for quantitative real-time PCR studies in the dentate gyrus after experimental febrile seizures.
abstract: BACKGROUND
  Quantitative real-time PCR (qPCR) is a commonly used technique to quantify gene expression levels. Validated normalization is essential to obtain reliable qPCR data. In that context, normalizing to multiple reference genes has become the most popular method. However, expression of reference genes may vary per tissue type, developmental stage and in response to experimental treatment. It is therefore imperative to determine stable reference genes for a specific sample set and experimental model. The present study was designed to validate potential reference genes in hippocampal tissue from rats that had experienced early-life febrile seizures (FS). To this end, we applied an established model in which FS were evoked by exposing 10-day old rat pups to heated air. One week later, we determined the expression stability of seven frequently used reference genes in the hippocampal dentate gyrus.
  
  
  RESULTS
  Gene expression stability of 18S rRNA, ActB, GusB, Arbp, Tbp, CycA and Rpl13A was tested using geNorm and Normfinder software. The ranking order of reference genes proposed by geNorm was not identical to that suggested by Normfinder. However, both algorithms indicated CycA, Rpl13A and Tbp as the most stable genes, whereas 18S rRNA and ActB were found to be the least stably expressed genes.
  
  
  CONCLUSIONS
  Our data demonstrate that the geometric averaging of at least CycA, Rpl13A and Tbp allows reliable interpretation of gene expression data in this experimental set-up. The results also show that ActB and 18S rRNA are not suited as reference genes in this model.
authors.affiliation: BIOMED Research Institute, Hasselt University and transnational University Limburg, Agoralaan Bld C, 3590, Diepenbeek, Belgium.
authors.forename: Ann
authors.initials: A
authors.lastname: Swijsen
authors.forename: Katherine
authors.initials: K
authors.lastname: Nelissen
authors.forename: Daniel
authors.initials: D
authors.lastname: Janssen
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Rigo
authors.forename: Govert
authors.initials: G
authors.lastname: Hoogland
mesh_terms: D000388:Air; D000465:Algorithms; D000818:Animals; D000831:Animals, Newborn; D021984:Cyclophilin A / Q000235:genetics*; D018891:Dentate Gyrus / Q000378:metabolism*; D015870:Gene Expression; D020869:Gene Expression Profiling; D020043:Genes, Essential; D006358:Hot Temperature; D008297:Male; D009419:Nerve Tissue Proteins / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D012269:Ribosomal Proteins / Q000235:genetics*; D003294:Seizures, Febrile / Q000235:genetics*; D012984:Software; D035181:TATA-Box Binding Protein / Q000235:genetics*
chemical_list: D009419:Nerve Tissue Proteins; D012269:Ribosomal Proteins; C489210:Rpl13a protein, rat; D035181:TATA-Box Binding Protein; D021984:Cyclophilin A