tissue: whole body extracts (30 organisms) age: 96h old effect concentration: 10% agent: 3,5-dichloroaniline
Treatment protocol
Two sets of structural analogues, defined as narcotics (alcohols: ethanol, methanol and isopropanol) and as polar narcotics (chlorinated anilines: aniline, 4-chloroaniline, 3,5-dichloroaniline and 2,3,4-trichloroaniline) were selected for short-term toxicity experiments. Daphnia magna neonates (between 0 and 24h old) were exposed for 96h in glass recipients to each chemicals’ respective EC1-96h and EC10-96h (determined based on 96h immobilization experiments) to allow multi-level effect assessment (growth, energy reserves and gene transcription was assessed) of different chemicals at the same level of acute toxicity (exposure at equitoxic acute concentration). The experiments were performed in triplicate at a density of 1daphnid/5ml of reconstituted fresh 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). Test media were renewed every 24h for the alcohols (to account for their high(er) volatility) and every 48h for the anilines. Organisms were fed every other day with a mixture of P. subcapitata and C. reinhardtii in a 3:1 ratio. The necessary amounts of organisms for the respective endpoints (growth and energy use) were collected after 48h and 96h of exposure and for gene transcription analyses after 96h of exposures. 30 pooled daphnids per replica were used for gene transcription analyses.
Extracted molecule
total RNA
Extraction protocol
Approximately 30 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 Vandenbrouck et al. (2009). 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, Cy5/Cy3 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 (30 organisms) age: 96h old sample type: reference
Treatment protocol
Two sets of structural analogues, defined as narcotics (alcohols: ethanol, methanol and isopropanol) and as polar narcotics (chlorinated anilines: aniline, 4-chloroaniline, 3,5-dichloroaniline and 2,3,4-trichloroaniline) were selected for short-term toxicity experiments. Daphnia magna neonates (between 0 and 24h old) were exposed for 96h in glass recipients to each chemicals’ respective EC1-96h and EC10-96h (determined based on 96h immobilization experiments) to allow multi-level effect assessment (growth, energy reserves and gene transcription was assessed) of different chemicals at the same level of acute toxicity (exposure at equitoxic acute concentration). The experiments were performed in triplicate at a density of 1daphnid/5ml of reconstituted fresh 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). Test media were renewed every 24h for the alcohols (to account for their high(er) volatility) and every 48h for the anilines. Organisms were fed every other day with a mixture of P. subcapitata and C. reinhardtii in a 3:1 ratio. The necessary amounts of organisms for the respective endpoints (growth and energy use) were collected after 48h and 96h of exposure and for gene transcription analyses after 96h of exposures. 30 pooled daphnids per replica were used for gene transcription analyses.
Extracted molecule
total RNA
Extraction protocol
Approximately 30 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 Vandenbrouck et al. (2009). 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, Cy5/Cy3 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. The hybridization design was a universal reference design (a mixture of aliquots from control and exposed samples), which is recommended when class discovery is the main purpose of the experiment. One of the three biological replicates of each exposure condition was labeled with one dye, the remaining two samples were labeled with the second dye.
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).
Data processing
Statistical analyses were performed using the R package limma (linear models for microarray data). Spots for which red and green median foreground intensity < median background intensity + 2SD on all arrays were deleted before analysis. Median intensity data was background corrected using a normal-exponential convolution model. Subsequently, normalization between-arrays was performed using Variance Stabilization Normalization (vsn). For each probe, a linear model was fitted to intensity ratios, after which probes were ranked in order of evidence of differential expression using an empirical Bayes method. Both high and low concentrations of each chemical stressor were contrasted against their respective controls. Genes with p < 0.05 and log2FC < -0.75 or log2FC > 0.75 (log2 fold change) were considered as differentially transcribed gene fragments.
