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VALIDATION OF REFERENCE GENES FOR REALREAL-TIME QUANTITATIVE
PCR IN DAPHNIA MAGNA FOLLOWING IBUPROFEN EXPOSURE
Lars H. Heckmann1,*, Richard Connon1, Thomas H. Hutchinson2, Steve J. Maund3, Richard M. Sibly1, Amanda Callaghan1
1The University of Reading, School of Biological Sciences, Environmental Biology, PO Box 228, RG6 6 AJ, UK; 2AstraZeneca
Global SHE, Brixham Environmental Laboratory, Devon, TQ5 8BA, UK; 3Syngenta Crop Protection AG, 4002 Basel, Switzerland ; *[email protected]
ABSTRACT
RESULTS AND DISCUSSION
Real-time quantitative PCR (QPCR) is a preferred technique
for measuring target gene expression. However, it relies on
accurate and relevant normalisation to invariant internal
controls such as reference genes (RG). Here we evaluate the
suitability of candidate RGs for QPCR in Daphnia magna
following exposure to ibuprofen (IB). The approach is
illustrated using as target gene the D. magna ortholog of
leukotriene B4 12-hydroxy-dehydrogenase (Ltb4dh), which
was identified as being up-regulated in a suppressive
subtractive hybridisation following 24 h exposure to 63-81
mg IB/l [1]. QPCR confirms that Ltb4dh is up-regulated in
response to ibuprofen exposure.
The geNorm software ranked the least variable genes as; UBC
= GAPDH < Act < WARS < SDH < TBP < 18S < 28S (see
Fig. 1 for full names). Alpha-tubulin and cyclophilin were
down-regulated by IB and so were not included in the
geNorm analysis, although this response warrants further
study. The optimal normalisation factor was NF3 which was
comprised of the geometric mean expression of UBC,
GAPDH and Act (Fig. 1). This NF was based on the lowest
recommended number of RGs with the lowest level of
variation [3]. The response to IB of target gene, Ltb4dh, was
little changed by application of NF3 (Fig. 2), although at the
highest concentration of IB, there was a slight increase in the
response, and a decrease of the variation between replicates.
Such reduction in the variation between replicates is exactly
what one hopes to achieve through normalisation, whereas
normalisation to more “noisy” RGs altered the outcome, in
some cases significantly (Fig. 3). QPCR confirmed that
Ltb4dh was up-regulated following exposure to IB [1].
NSAIDs are known to inhibit the biosynthesis of various
eicosanoids that play important regulatory and signalling
functions, e.g. regulation of ion flux [5]. In mammals, Ltb4dh
is involved in the metabolism of leukotriene B4 an eicosanoid
that is formed in the lipoxygenase pathway. There is evidence
of eicosanoid pathways in invertebrates being similar to the
mammalian pathways [5]. Further experimentation, involving
a global expression profile based on cDNA microarrays, is
underway to support this hypothesis and reveal the overall
molecular stress response of D. magna to IB.
INTRODUCTION
Transcriptomic techniques are now regularly applied in
ecotoxicology to measure the impact of stressors and develop
an understanding of the mechanisms of toxicity. The accuracy
of these techniques relies on normalisation to an invariant
reference gene (RG) or other internal control. Unfortunately,
the expression of RGs can vary between tissues and
experimental conditions, which makes it crucial to validate
them prior to application [2].
Here, we use the geNorm algorithm [3] to estimate the
variability of 10 potential RGs for QPCR in Daphnia magna
following a 24 h exposure to the non-steroidal antiinflammatory drug (NSAID) ibuprofen.
Figure 1. Pairwise variation of sequential norma-lisation
factors (NFn vs. NFn+1) estimated by geNorm. NF2 is based
Figure 2. Relative expression of target gene Ltb4dh in D.
magna following a 24 h exposure to ibuprofen. Mean relative
expression (n = 3) ± SD of target gene Ltb4dh without normalisation
(white bars) or with normalisation (black bars) to normalisation factor NF3
based on the geometric mean of reference genes UBC, GAPDH and Act.
An asterisk (*) indicates a significant difference (P<0.05) between the
highest treatment and any other treatment.
on the geometric mean of glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) and ubiquitin conjuga-ting enzyme (UBC); NF3 is NF2 and
actin (Act); NF4 is NF3 and tryptophanyl-tRNA synthetase (WARS); NF5
is NF4 and succinate dehydrogenase (SDH); NF6 is NF5 and TATA-box
binding protein (TBP); NF7 is NF6 and 28S rRNA (28S); NF8 is NF7 and
18S rRNA (18S).
MATERIALS AND METHODS
• The experiment consisted of triplicates of a control and 3 ibuprofensodium (CAS no. 31121-93-4) treatments, here referred to as the free base
concentration. 100 D. magna (<24 h) were exposed 24 h to 0, 20, 40 and
80 mg IB/l. Total RNA was extracted using the RNeasy Mini kit
(Qiagen), and subsequently reverse transcribed using the Omniscript RT
kit (Qiagen).
• QPCR was conducted on the GeneAmp 5700 SDS (Applied
Biosystems) using ABsoluteTM QPCR SYBR® Green ROX (500 nM) mix
(ABgene). Relative expression of the RGs, calculated by DART-PCR [4],
was exported as an Excel workbook (Microsoft) to geNorm [3]. The
geNorm algorithm calculates the individual gene variability.
Figure 3. Relative expression of target gene Ltb4dh in D.
magna following a 24 h exposure to ibuprofen. Mean relative
expression (n = 3) ± SD of Ltb4dh following normalisation to 10
candidate reference genes and a normalisation factor (NF). For
comparison, the normalised relative expression of Ltb4dh is displayed by
treatment group. Relative expression of Ltb4dh normalised to: Act (1);
alpha-tubulin (2); cyclophilin (3); GAPDH (4); SDH (5); TBP (6); WARS
(7); UBC (8); 18S (9); 28S (10); NF3 based on the geometric mean of
UBC, GAPDH and Act (11); and Ltb4dh without normalisation (12). An
asterisk (*) indicates a signifi-cant difference (P<0.05) from Ltb4dh
normalised to NF3.
REFERENCES – 1. Heckmann et al., 2005. SETAC Europe 15th Annual Meeting; 2. Huggett
et al., 2005. Genes and Immunity 6:279-284; 3. Vandesompele et al., 2002. Genome Biology
3:research0034; 4. Peirson et al., 2003. Nucleic Acids Research 31:e73; 5. Rowley et al., 2005.
Journal of Experimental Biology 208:3-14.
CONCLUSION
No gene can be assumed a priori to be unaffected by
ibuprofen, so validation of reference genes is essential for
QPCR. Here we identify an unbiased normalisation factor
based on three relatively invariant reference genes: UBC,
GAPDH and Act.