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|Title: ||Validation of reference genes for quantitative real-time PCR studies in the dentate gyrus after experimental febrile seizures.|
|Authors: ||Swijsen, Ann|
|Issue Date: ||2012|
|Citation: ||BMC Research Notes, 5 (Article 685), p. 1-8|
|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.|
|Type: ||Journal Contribution|
|Appears in Collections: ||Research publications|
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