Background: Transcriptomic techniques are now being applied in ecotoxicology and toxicology to measure the impact of stressors and develop understanding of mechanisms of toxicity. Microarray technology in particular offers the potential to measure thousands of gene responses simultaneously. However, it is important that microarrays responses should be validated, at least initially, using real-time quantitative polymerase chain reaction (QPCR). The accurate measurement of target gene expression requires normalisation to an invariant internal control e.g., total RNA or reference genes. Reference genes are preferable, as they control for variation inherent in the cDNA synthesis and PCR. However, reference gene expression can vary between tissues and experimental conditions, which makes it crucial to validate them prior to application. Results: We evaluated 10 candidate reference genes for QPCR in Daphnia magna following a 24 h exposure to the non-steroidal anti-inflammatory drug (NSAID) ibuprofen (IB) at 0, 20, 40 and 80 mg IB l-1. Six of the 10 candidates appeared suitable for use as reference genes. As a robust approach, we used a combination normalisation factor (NF), calculated using the geNorm application, based on the geometric mean of three selected reference genes: glyceraldehyde-3-phosphate dehydrogenase, ubiquitin conjugating enzyme and actin. The effects of normalisation are illustrated using as target gene leukotriene B4 12-hydroxydehydrogenase (Ltb4dh), which was up-regulated following 24 h exposure to 63-81 mg IB l-1. Conclusions: As anticipated, use of the NF clarified the response of Ltb4dh in daphnids exposed to sublethal levels of ibuprofen. Our findings emphasise the importance in toxicogenomics of finding and applying invariant internal QPCR control(s) relevant to the study conditions.
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