Abstract
BackgroundThe planktonic microcrustacean Daphnia pulex is among the best-studied animals in ecological, toxicological and evolutionary research. One aspect that has sustained interest in the study system is the ability of D. pulex to develop inducible defence structures when exposed to predators, such as the phantom midge larvae Chaoborus. The available draft genome sequence for D. pulex is accelerating research to identify genes that confer plastic phenotypes that are regularly cued by environmental stimuli. Yet for quantifying gene expression levels, no experimentally validated set of internal control genes exists for the accurate normalization of qRT-PCR data.ResultsIn this study, we tested six candidate reference genes for normalizing transcription levels of D. pulex genes; alpha tubulin (aTub), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box binding protein (Tbp) syntaxin 16 (Stx16), X-box binding protein 1 (Xbp1) and CAPON, a protein associated with the neuronal nitric oxide synthase, were selected on the basis of an earlier study and from microarray studies. One additional gene, a matrix metalloproteinase (MMP), was tested to validate its transcriptional response to Chaoborus, which was earlier observed in a microarray study. The transcription profiles of these seven genes were assessed by qRT-PCR from RNA of juvenile D. pulex that showed induced defences in comparison to untreated control animals. We tested the individual suitability of genes for expression normalization using the programs geNorm, NormFinder and BestKeeper. Intriguingly, Xbp1, Tbp, CAPON and Stx16 were selected as ideal reference genes. Analyses on the relative expression level using the software REST showed that both classical housekeeping candidate genes (aTub and GAPDH) were significantly downregulated, whereas the MMP gene was shown to be significantly upregulated, as predicted. aTub is a particularly ill suited reference gene because five copies are found in the D. pulex genome sequence. When applying aTub for expression normalization Xbp1 and Tbp are falsely reported as significantly upregulated.ConclusionsOur results suggest that the genes Xbp1, Tbp, CAPON and Stx16 are suitable reference genes for accurate normalization in qRT-PCR studies using Chaoborus-induced D. pulex specimens. Furthermore, our study underscores the importance of verifying the expression stability of putative reference genes for normalization of expression levels.
Highlights
The planktonic microcrustacean Daphnia pulex is among the best-studied animals in ecological, toxicological and evolutionary research
Normalization is accomplished in several ways; the expression values from target genes of interest can be balanced against the total amount of RNA in the reactions, balanced against synthetic RNA that is "spiked" in each reaction, or measured against internal reference genes
To improve robustness of the experiment, it is recommended to use more than one reference gene [7,9] and to verify that their transcriptional activity are stable across conditions and tissue types
Summary
The planktonic microcrustacean Daphnia pulex is among the best-studied animals in ecological, toxicological and evolutionary research. For quantifying gene expression levels, no experimentally validated set of internal control genes exists for the accurate normalization of qRT-PCR data. Normalization is accomplished in several ways; the expression values from target genes of interest can be balanced against the total amount of RNA in the reactions, balanced against synthetic RNA that is "spiked" in each reaction, or measured against internal reference genes. This last method is generally considered reliable and is frequently applied for quantifying relative gene expression [7]. In samples with different amounts of input RNA the ratio of two ideal reference genes remains constant
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