Open Access Highly Accessed Research article

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

Muriel E de Boer1*, Tjalf E de Boer1, Janine Mariën1, Martijn JTN Timmermans12, Benjamin Nota1, Nico M van Straalen1, Jacintha Ellers1 and Dick Roelofs1

Author Affiliations

1 Institute of Ecological Science, VU University Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands

2 Current address: Faculty of Natural Sciences, Department of Ecology & Evolution, Imperial College London Exhibition Road, London SW7 2AZ, UK

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BMC Molecular Biology 2009, 10:54  doi:10.1186/1471-2199-10-54

Published: 1 June 2009

Abstract

Background

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, Folsomia candida and Orchesella cincta. Treatments for F. candida were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for O. cincta cadmium, desiccation, heat shock and starvation.

Results

Eight potential reference genes for F. candida and seven for O. cincta were ranked by their stability per stress factor using the programs geNorm and Normfinder. For F. candida the succinate dehydrogenase (SDHA) and eukaryotic transcription initiation factor 1A (ETIF) genes were found the most stable over the different treatments, while for O. cincta, the beta actin (ACTb) and tyrosine 3-monooxygenase (YWHAZ) genes were the most stable.

Conclusion

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.