Reference genes for QRT-PCR tested under various stress conditions in Folsomia candida and Orchesella cincta (Insecta, Collembola)

<p>Abstract</p> <p>Background</p> <p>Genomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology. To investigate a small to medium number of genes or to confirm large scale microarray studies, Quantitative Reverse Transcriptase PCR (QRT-PCR) can achieve high accuracy of quantification when key standards, such as normalization, are carefully set. In this study, we validated potential reference genes for their use as endogenous controls under different chemical and physical stresses in two species of soil-living Collembola, <it>Folsomia candida </it>and <it>Orchesella cincta</it>. Treatments for <it>F. candida </it>were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for <it>O. cincta </it>cadmium, desiccation, heat shock and starvation.</p> <p>Results</p> <p>Eight potential reference genes for <it>F. candida </it>and seven for <it>O. cincta </it>were ranked by their stability per stress factor using the programs geNorm and Normfinder. For <it>F. candida </it>the succinate dehydrogenase (<it>SDHA</it>) and eukaryotic transcription initiation factor 1A (<it>ETIF</it>) genes were found the most stable over the different treatments, while for <it>O. cincta</it>, the beta actin (<it>ACTb</it>) and tyrosine 3-monooxygenase (<it>YWHAZ</it>) genes were the most stable.</p> <p>Conclusion</p> <p>We present a panel of reference genes for two emerging ecological genomic model species tested under a variety of treatments. Within each species, different treatments resulted in differences in the top stable reference genes. Moreover, the two species differed in suitable reference genes even when exposed to similar stresses. This might be attributed to dissimilarity of physiology. It is vital to rigorously test a panel of reference genes for each species and treatment, in advance of relative quantification of QRT-PCR gene expression measurements.</p

Collembase: a repository for springtail genomics and soil quality assessment

<p>Abstract</p> <p>Background</p> <p>Environmental quality assessment is traditionally based on responses of reproduction and survival of indicator organisms. For soil assessment the springtail <it>Folsomia candida </it>(Collembola) is an accepted standard test organism. We argue that environmental quality assessment using gene expression profiles of indicator organisms exposed to test substrates is more sensitive, more toxicant specific and significantly faster than current risk assessment methods. To apply this species as a genomic model for soil quality testing we conducted an EST sequencing project and developed an online database.</p> <p>Description</p> <p>Collembase is a web-accessible database comprising springtail (<it>F. candida</it>) genomic data. Presently, the database contains information on 8686 ESTs that are assembled into 5952 unique gene objects. Of those gene objects ~40% showed homology to other protein sequences available in GenBank (blastx analysis; non-redundant (nr) database; expect-value < 10^-5). Software was applied to infer protein sequences. The putative peptides, which had an average length of 115 amino-acids (ranging between 23 and 440) were annotated with Gene Ontology (GO) terms. In total 1025 peptides (~17% of the gene objects) were assigned at least one GO term (expect-value < 10^-25). Within Collembase searches can be conducted based on BLAST and GO annotation, cluster name or using a BLAST server. The system furthermore enables easy sequence retrieval for functional genomic and Quantitative-PCR experiments. Sequences are submitted to GenBank (Accession numbers: <ext-link ext-link-type="gen" ext-link-id="EV473060">EV473060</ext-link> – <ext-link ext-link-type="gen" ext-link-id="EV481745">EV481745</ext-link>).</p> <p>Conclusion</p> <p>Collembase <url>http://www.collembase.org</url> is a resource of sequence data on the springtail <it>F. candida</it>. The information within the database will be linked to a custom made microarray, based on the Agilent platform, which can be applied for soil quality testing. In addition, Collembase supplies information that is valuable for related scientific disciplines such as molecular ecology, ecogenomics, molecular evolution and phylogenetics.</p

Venn-diagram showing the cluster overlap between the three libraries for the total dataset: Cad: cadmium enriched library; Phe: phenanthrene enriched library; Nor: normalized library

<p><b>Copyright information:</b></p><p>Taken from "Collembase: a repository for springtail genomics and soil quality assessment"</p><p>http://www.biomedcentral.com/1471-2164/8/341</p><p>BMC Genomics 2007;8():341-341.</p><p>Published online 27 Sep 2007</p><p>PMCID:PMC2234260.</p><p></p

Relative abundance of six cDNAs before (upper) and after (lower) normalization as measured using quantitative PCR

<p><b>Copyright information:</b></p><p>Taken from "Collembase: a repository for springtail genomics and soil quality assessment"</p><p>http://www.biomedcentral.com/1471-2164/8/341</p><p>BMC Genomics 2007;8():341-341.</p><p>Published online 27 Sep 2007</p><p>PMCID:PMC2234260.</p><p></p> Act: β-actin; 28S: 28S rDNA; De: RNA helicase Dead1; RXR: RXR-USP; Ub: Ultrabithorax; Kr: Kruppel