Abstract
BackgroundQuantitative 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.ResultsGene 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.ConclusionsOur 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.
Highlights
Quantitative real-time PCR is a commonly used technique to quantify gene expression levels
Cycle threshold values of candidate reference genes When all samples were taken together, the reference genes showed cycle threshold (Cq) values varying from a Cq value of 15.40 for Acidic ribosomal phosphoprotein P0 (Arbp) to 31.22 for Tbp (Additional file 1)
With a Cq ≤ 22.23, Arbp, ActB, CycA and Rpl13A showed lower Cq values than GusB and Tbp that had a Cq ≥ 24.42. 18S 18S subunit ribosomal RNA (rRNA) displayed the highest Cq variability, ranging from 17.91 to 29.75
Summary
Quantitative real-time PCR (qPCR) is a commonly used technique to quantify gene expression levels. 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. Quantitative real-time PCR (qPCR) is a commonly used tool to quantify gene expression. An advantage of this highly specific and sensitive technique is that it allows analysis on small amounts of starting material [7,8]. It has been shown that the accuracy of qPCR data further improves when at least three reference genes are used for normalization [9]
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