zebrafish embryos (48 hpf) exposed to control medium directly upon fertilization (hpf = hours post fertilization)
Treatment protocol
Collection of zebrafish eggs and exposure of embryos were performed according to established protocols (Nagel, 1998; Schulte and Nagel, 1994). Control treatment was performed in 10 ml test medium (2 mM CaCl2, 0.5 mM MgSO4, 0.75 mM NaHCO3, 0.08 mM KCl) in triplicates starting on three different days. The concentration used for microarray analysis corresponded to the LOEC (lowest observed effect concentration) for survival (12.4 µM) of the embryo test. 100 zebrafish embryos were exposed from two hours post fertilization (hpf) for up to 48 h hpf. Control embryos were exposed in 10 ml test medium.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from zebrafish embryos using Trizol Reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer`s instructions. Contaminations of genomic DNA were removed by treatment with DNAse I (Roche, Grenzach, Germany) for 15 min at 25° C.
Label
cy5
Label protocol
For indirect labelling of cDNA to the cyanine dyes, a reverse transcriptase labelling mixture including aminoallyl-UTP was added to the RNA. The reaction was incubated at 42°C for 14 hours to generate aminoallyl-labelled cDNA. Unincorporated aa-dUTP and free amines were removed using the QIAquick PCR purification kit (Qiagen, Hilden, Germany). The samples were dried in a vacuum centrifuge. Aminoallyl-labelled cDNA was resuspended in 4.5 µl 0.1 M sodium carbonate buffer (pH 9.0) and mixed with 4,5 µl of Cy3 or Cy5 monoreactive dye (Amersham Biosciences, Munich, Germany) in DMSO. Dye coupling was performed for one hour at room temperature in the dark. Uncoupled dyes were removed using the QIAquick PCR purification kit (Qiagen).
Channel 2
Source name
zebrafish embryos (48 hpf) exposed to 3,4-dichloroaniline, test 3, cy3-labeled
zebrafish embryos (48 hpf) exposed to control medium directly upon fertilization (hpf = hours post fertilization)
Treatment protocol
Collection of zebrafish eggs and exposure of embryos were performed according to established protocols (Nagel, 1998; Schulte and Nagel, 1994). Exposure was performed in 10 ml test medium (2 mM CaCl2, 0.5 mM MgSO4, 0.75 mM NaHCO3, 0.08 mM KCl) in triplicates starting on three different days. The concentration used for microarray analysis corresponded to the LOEC (lowest observed effect concentration) for survival (12.4 µM) of the embryo test. 100 zebrafish embryos were exposed from two hours post fertilization (hpf) for up to 48 h hpf. Control embryos were exposed in 10 ml test medium.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from zebrafish embryos using Trizol Reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer`s instructions. Contaminations of genomic DNA were removed by treatment with DNAse I (Roche, Grenzach, Germany) for 15 min at 25° C
Label
cy3
Label protocol
For indirect labelling of cDNA to the cyanine dyes, a reverse transcriptase labelling mixture including aminoallyl-UTP was added to the RNA. The reaction was incubated at 42°C for 14 hours to generate aminoallyl-labelled cDNA. Unincorporated aa-dUTP and free amines were removed using the QIAquick PCR purification kit (Qiagen, Hilden, Germany). The samples were dried in a vacuum centrifuge. Aminoallyl-labelled cDNA was resuspended in 4.5 µl 0.1 M sodium carbonate buffer (pH 9.0) and mixed with 4,5 µl of Cy3 or Cy5 monoreactive dye (Amersham Biosciences, Munich, Germany) in DMSO. Dye coupling was performed for one hour at room temperature in the dark. Uncoupled dyes were removed using the QIAquick PCR purification kit (Qiagen).
Hybridization protocol
The two labelling reactions were combined, vacuum dried and resuspended in 32 µl hybridisation buffer (50% formamide, 5x SSC, 0.1% SDS and 0.1 mg/ml salmon sperm DNA). The samples were denaturised by heating to 95°C for 3 minutes. A volume of 30 µl of the reaction were hybridised to the 14 k zebrafish oligonucleotide slides. The slides were incubated for 14 hours at 42°C in a water bath. Washing was performed in 0.1% SDS, 2x SSC for 10 minutes, followed by three additional washes for 10 minutes in 0.2x SSC, 0.1x SSC and 0.05x SSC at room temperature. The slides were dried by centrifugation. Microarray hybridisations were performed for all replicates of samples treated with 12.4 µM 3,4-DCA (n = 3). For every single experiment, dye-swaps were performed.
Scan protocol
The hybridised slides were scanned at 10 µm resolution using the ScanArray Express Scanner (Perkin Elmer, USA) at 532 nm (Cy3) and 635 nm (Cy5). The laser power of the scanner was adjusted between 80% to 100% and PMT (photo multiplier value) was preset between 60 and 80, depending on the amount of background fluorescence and the saturation of signals.
Description
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Data processing
Microarray data were analyzed using the TM4 software package (http://www.tm4.org/) (Saeed et al., 2003). TIGR Spotfinder 3.0.0 beta was used to identify spots on the array and to assess the quality of spots for downstream analyses. TIGR MIDAS 2.19 was then used to adjust the data by applying LOWESS normalization (Quackenbush, 2002). A 30% quantile was used to remove data with weak fluorescence recordings for both Cy3 and Cy5 prior to subsequent statistical analyses. Genes with significantly altered expression patterns were identified by one-class SAM (significance analysis of microarrays) (Tusher et al., 2001).
