tissue: whole body extracts (40 organisms) age: 96h old
Treatment protocol
The organisms were exposed to two PAHs, fluoranthene and pyrene (Sigma-Aldrich, Belgium) both as single compounds and as binary mixtures. EC50 (immobility at 48h) values of the single compounds (when fed) were established to set the concentrations of the individual component exposures equal to 0; 0.062; 0.125 and 0.250 Toxic Units (TU) when one TU is defined as the EC50 (immobility at 48h) of the single compound. For the binary mixtures, concentrations for a total of 6 (to 8) combinations were determined based on a fixed ratio design. During the 96h exposure experiments, 40 Daphnia/replica were kept at a density of 1 organism/10mL OECD standard water (CaCl2.2H2O, 2mM; MgSO4.7H2O, 500μM; NaHCO3, 771μM; KCl, 77.1μM; water hardness, 250 mg CaCO3; pH 7.8; OECD guideline 203, annex 2). Since fluoranthene and pyrene are both poorly soluble in water, a solvent was used. The PAHs were dissolved in acetone; final solvent concentrations of 0.01% were used. Therefore besides the pure OECD water control, an additional solvent control sample was included in each test. Organisms were fed every other day – when test solutions were renewed - with a mixture of P. subcapitata and C. reinhardtii in a 3:1 ratio.
Extracted molecule
total RNA
Extraction protocol
Approximately 40 Daphnia/replicate were collected. Samples were shock-frozen in liquid nitrogen and stored in RNAlater (Ambion, USA) at -80°C. All RNA extractions were performed using the TRIzol® method (Invitrogen, Belgium) followed by a DNAse treatment using 1U RNAse-free DNAse and 1U RNAse inhibitor (Fermentas, Germany) per 30µl sample and subsequent phenol/chloroform extractions. The purity of the RNA samples was checked using the ND-1000 spectrophotometer (Nanodrop®, USA) through measurement of the 260nm/280nm and 260nm/230nm absorbance. For all used samples these ratios were respectively above 1.90 and 2.10. To verify the intactness of the RNA samples, a denaturating formaldehyde agarose gel electrophoresis was performed to visualize the 18S and 28S ribosomal bands.
Label
Cy5
Label protocol
The production of cDNA and subsequent amino-allyl labelling were performed according to the protocol described in detail by Van der Ven et al. (2005) and Soetaert et al. (2006). In brief, 5µg of total RNA was mixed with Lucidea control mRNA spike mix (Amersham, UK) to be reverse-transcribed using the Superscript II, Random hexamer primers (both Invitrogen) in the presence of dNTPs with 2/3 aa-dTTP/dTTP (Sigma-Aldrich, Belgium). After an overnight incubation period (42°C) the amino-allyl-incorporated cDNA was purified using a modified Qiagen PCR spin colomn protocol (Van der Ven et al., 2005). In a next step, Cy3/Cy5 esters (Amersham) were used for covalent coupling to the amino-allyl labelled cDNA. The vacuum dried cDNA samples (dissolved in a 0,1M carbonate buffer (pH 9.0)) were mixed with the Cy3/Cy5 esters (dissolved in 100% DMSO) and incubated for an hour in the dark at room temperature. Hereafter, a second cleanup reaction was performed to establish the removal of the remaining uncoupled dyes (QIAquick PCR purification kit, Qiagen, USA). The quality of eluted and labelled cDNA was analysed using the Nanodrop® spectrophotometer. Samples with an FOI (frequence of incorporated dye) between 20 and 50 were selected for hybridization and an amount of 50 pmol labelled target was vacuum dried.
tissue: whole body extracts (40 organisms) age: 96h old
Treatment protocol
The organisms were exposed to two PAHs, fluoranthene and pyrene (Sigma-Aldrich, Belgium) both as single compounds and as binary mixtures. EC50 (immobility at 48h) values of the single compounds (when fed) were established to set the concentrations of the individual component exposures equal to 0; 0.062; 0.125 and 0.250 Toxic Units (TU) when one TU is defined as the EC50 (immobility at 48h) of the single compound. For the binary mixtures, concentrations for a total of 6 (to 8) combinations were determined based on a fixed ratio design. During the 96h exposure experiments, 40 Daphnia/replica were kept at a density of 1 organism/10mL OECD standard water (CaCl2.2H2O, 2mM; MgSO4.7H2O, 500μM; NaHCO3, 771μM; KCl, 77.1μM; water hardness, 250 mg CaCO3; pH 7.8; OECD guideline 203, annex 2). Since fluoranthene and pyrene are both poorly soluble in water, a solvent was used. The PAHs were dissolved in acetone; final solvent concentrations of 0.01% were used. Therefore besides the pure OECD water control, an additional solvent control sample was included in each test. Organisms were fed every other day – when test solutions were renewed - with a mixture of P. subcapitata and C. reinhardtii in a 3:1 ratio.
Extracted molecule
total RNA
Extraction protocol
Approximately 40 Daphnia/replicate were collected. Samples were shock-frozen in liquid nitrogen and stored in RNAlater (Ambion, USA) at -80°C. All RNA extractions were performed using the TRIzol® method (Invitrogen, Belgium) followed by a DNAse treatment using 1U RNAse-free DNAse and 1U RNAse inhibitor (Fermentas, Germany) per 30µl sample and subsequent phenol/chloroform extractions. The purity of the RNA samples was checked using the ND-1000 spectrophotometer (Nanodrop®, USA) through measurement of the 260nm/280nm and 260nm/230nm absorbance. For all used samples these ratios were respectively above 1.90 and 2.10. To verify the intactness of the RNA samples, a denaturating formaldehyde agarose gel electrophoresis was performed to visualize the 18S and 28S ribosomal bands.
Label
Cy3
Label protocol
The production of cDNA and subsequent amino-allyl labelling were performed according to the protocol described in detail by Van der Ven et al. (2005) and Soetaert et al. (2006). In brief, 5µg of total RNA was mixed with Lucidea control mRNA spike mix (Amersham, UK) to be reverse-transcribed using the Superscript II, Random hexamer primers (both Invitrogen) in the presence of dNTPs with 2/3 aa-dTTP/dTTP (Sigma-Aldrich, Belgium). After an overnight incubation period (42°C) the amino-allyl-incorporated cDNA was purified using a modified Qiagen PCR spin colomn protocol (Van der Ven et al., 2005). In a next step, Cy3/Cy5 esters (Amersham) were used for covalent coupling to the amino-allyl labelled cDNA. The vacuum dried cDNA samples (dissolved in a 0,1M carbonate buffer (pH 9.0)) were mixed with the Cy3/Cy5 esters (dissolved in 100% DMSO) and incubated for an hour in the dark at room temperature. Hereafter, a second cleanup reaction was performed to establish the removal of the remaining uncoupled dyes (QIAquick PCR purification kit, Qiagen, USA). The quality of eluted and labelled cDNA was analysed using the Nanodrop® spectrophotometer. Samples with an FOI (frequence of incorporated dye) between 20 and 50 were selected for hybridization and an amount of 50 pmol labelled target was vacuum dried.
Hybridization protocol
Prior to hybridization, arrays were incubated for 30-45 min at 42°C in a prehybridization solution (50% formamide, 5x saline sodium citrate (SSC), 0.1% sodium dodecyl sulphate (SDS), 0.1mg/mL BSA). The vacuum dried targets (both reference and treatment target) were resuspended in a hybridization solution (50% formamide, 5x SSC, 0.1% SDS, 0.1mg/mL BSA and 0.1mg/mL sheared salmon sperm) and denaturated at 95°C. The cooled targets were subsequently applied onto the prehybridized slides and incubated overnight at 42°C. After hybridization, arrays were washed in solutions with increasing stringency (decreasing concentrations of SSC and SDS) and dried with N2.
Scan protocol
Scanning and analysing of the slides was performed using the Genepix personal 4100 Scanner and Genepix pro Software (both Axon instruments, USA). Cy3 and Cy5 fluorescent signals were scanned at respectively 532 and 635nm and the PMT (photomultiplier tube) values were adjusted to reach a ratio (Cy5/Cy3) around 1. Spots were identified and ratio’s quantified by means of the Genepix software 5.0 (Axon Instruments).
Description
Hybridization: Mixture_0_062TUFlu_0_185TUPyr versus Reference sample
Data processing
Prior to the actual statistical analysis, the quality of the microarray spots was assessed. Over-saturated spots and bad quality spots identified during the feature identification process (visual inspection of the slides) were excluded from further analysis. Hereafter, a local background correction was performed on these datasets (BASE). Those spots with foreground levels lower than the background levels for both channels were excluded from the analysis. Spots for which the foreground was smaller than the mean (local) background ±2 standard deviations for both channels were given a ‘weight’ of 0.1. All other included spots are given a weight of 1. Subsequently, microarray datasets were normalized using the variance stabilizing normalization (VSN) (Huber 2002). The significantly differentially expressed genes (between control and treatment) were statistically determined using LIMMA (LInear Model for Microarray Analysis) (Smyth 2004). Results were corrected for multiple testing using the false discovery rate (FDR) and differential gene expression was detected in probes with p<0.05. An additional threshold which was taken into account is the fold induction cut-off (Log2) of (-)0.85. In conclusion, genes with fold inductions outside these thresholds and p-values below 0.05 were selected as differentially expressed.
