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1.  Comments on sequence normalization of tiling array expression 
Bioinformatics  2009;25(17):2171-2173.
Motivation: Methods to improve tiling array expression signals are needed to accurately detect genome features. Royce et al. provide statistical normalizations of tile signal based on probe sequence content that promises improved accuracy, and should be independently verified.
Results: Assessment of the sequence content normalization methods identified a problem: confounding of probe sequence content with gene structure (intron/exon) sequence content. Normalization obscured tile signal changes at gene structure boundaries. This and other evidence suggests that simple sequence normalization does not improve detection of genes from tile expression data.
PMCID: PMC2800354  PMID: 19578171
2.  Predator-induced defences in Daphnia pulex: Selection and evaluation of internal reference genes for gene expression studies with real-time PCR 
BMC Molecular Biology  2010;11:50.
The 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.
In 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.
Our 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.
PMCID: PMC3148505  PMID: 20587017
3.  wFleaBase: the Daphnia genome database 
BMC Bioinformatics  2005;6:45.
wFleaBase is a database with the necessary infrastructure to curate, archive and share genetic, molecular and functional genomic data and protocols for an emerging model organism, the microcrustacean Daphnia. Commonly known as the water-flea, Daphnia's ecological merit is unequaled among metazoans, largely because of its sentinel role within freshwater ecosystems and over 200 years of biological investigations. By consequence, the Daphnia Genomics Consortium (DGC) has launched an interdisciplinary research program to create the resources needed to study genes that affect ecological and evolutionary success in natural environments.
These tools include the genome database wFleaBase, which currently contains functions to search and extract information from expressed sequenced tags, genome survey sequences and full genome sequencing projects. This new database is built primarily from core components of the Generic Model Organism Database project, and related bioinformatics tools.
Over the coming year, preliminary genetic maps and the nearly complete genomic sequence of Daphnia pulex will be integrated into wFleaBase, including gene predictions and ortholog assignments based on sequence similarities with eukaryote genes of known function. wFleaBase aims to serve a large ecological and evolutionary research community. Our challenge is to rapidly expand its content and to ultimately integrate genetic and functional genomic information with population-level responses to environmental challenges. URL: .
PMCID: PMC555599  PMID: 15752432

Results 1-3 (3)