strain: AB, ABO, and Tübingen genotype: wild type age: 96 hours post-fertilization developmental stage: embryo tissue: whole body treatment: sediment extract from Ehingen
Biomaterial provider
Fish Facility at the Institute of Toxicology and Genetics, Karlsruhe Institute of Technology
Treatment protocol
Zebrafish wild type strains AB, ABO and Tuebingen were kept and bred. 400 embryos were exposed for each treatment (negative control, sediment extract). Fertilized eggs were collected after spawning and exposed for 96 hpf at 27 °C for each treatment. Concentrations of the sediment extracts were selected as 10 mg sediment equivalent (SEQ) per ml test medium for the site Ehingen in a solvent concentration of 0.05 % DMSO (vol/vol). Embryos were grown in embryo medium (60 μg/ml Instant Ocean, Red Sea, Houston, TX; pH 6.73, Ca 0.8 mg/l; K 0.6 mg/l; Mg 2 mg/l; Na 16 mg/l; S 2 mg/l). Each treatment and control was performed in triplicates; each represents a full biological replicate.
Growth protocol
As described in Westerfield, 1993.
Extracted molecule
total RNA
Extraction protocol
1) Total RNA was isolated using the Nucleospin RNA L Kit (Macherey-Nagel, Dueren, Germany). 2) mRNA was extracted with the Ambion Purist Kit (Austin, TX).
Label
Cy5,Cy3
Label protocol
Labeled cDNA was synthesized from 1-2 ug mRNA using the Amersham direct cDNA labeling kit (Amersham Europe, Freiburg, Germany).
Channel 2
Source name
mRNA from zebrafish embryos NOT exposed to sediment extract from Ehingen, Germany
strain: AB, ABO, and Tübingen genotype: wild type age: 96 hours post-fertilization developmental stage: embryo tissue: whole body treatment: negative control
Biomaterial provider
Fish Facility at the Institute of Toxicology and Genetics, Karlsruhe Institute of Technology
Treatment protocol
Zebrafish wild type strains AB, ABO and Tuebingen were kept and bred. 400 embryos were exposed for each treatment (negative control, sediment extract). Fertilized eggs were collected after spawning and exposed for 96 hpf at 27 °C for each treatment. Concentrations of the sediment extracts were selected as 10 mg sediment equivalent (SEQ) per ml test medium for the site Ehingen in a solvent concentration of 0.05 % DMSO (vol/vol). Embryos were grown in embryo medium (60 μg/ml Instant Ocean, Red Sea, Houston, TX; pH 6.73, Ca 0.8 mg/l; K 0.6 mg/l; Mg 2 mg/l; Na 16 mg/l; S 2 mg/l). Each treatment and control was performed in triplicates; each represents a full biological replicate.
Growth protocol
As described in Westerfield, 1993.
Extracted molecule
total RNA
Extraction protocol
1) Total RNA was isolated using the Nucleospin RNA L Kit (Macherey-Nagel, Dueren, Germany). 2) mRNA was extracted with the Ambion Purist Kit (Austin, TX).
Label
Cy3,Cy5
Label protocol
Labeled cDNA was synthesized from 1-2 ug mRNA using the Amersham direct cDNA labeling kit (Amersham Europe, Freiburg, Germany).
Hybridization protocol
1) Removal of unincorporated nucleotides over Microcon 30 spin columns (Millipore, Bedford, MA). 2) The concentrated probes were hybridized to the microarray in 1xDIG Easy-Hyb buffer (Hoffmann-La Roche, Basel, CH) overnight at 42 C. 3) Cover slips were removed from the slides by flushing with 4xSSC. 4) Slides were washed in pre-warmed wash buffer 1 (2xSSC, 0.1% SDS) for 5 minutes at 42 C. 5) Washing in buffer 2 (0.1xSSC, 0.1% SDS) for 10 minutes at room temperature. 6) Washing in 0.1xSSC four times for 1 min at room temperature. 7) The slides were briefly dipped into 0.01xSSC at room temperature. 8) Centrifugation for 7 min at 800 rpm in an Eppendorf 5810R centrifuge.
Scan protocol
1) Scanned on a GenePix 4000B dual-laser scanner from Molecular Devices. 2) Both channels (532 nm for Cy3, and 635 nm for Cy5) were scanned in parallel. 3) Data was stored as 16-bit TIFF files. 4) The channels for Cy3 and Cy5 were balanced in each scan for approximately the same intensity profile. 5) Each array was scanned three times (low, medium and high scan) with different signal amplification factors (voltage settings of the photomultiplier tubes), but with the same laser power. For the low scan, no spot was saturated; in the high scan, the signal amplification for Cy5 was set to approximately 80% of maximum and Cy3 amplification was adjusted to this. The settings used in the medium scan lie between the low and the high scan. The absolute intensity values span the range from 0 to 65536. Signal intensities from low, medium, and high scans were mapped onto the same scale by an affine transformation. Transformation parameters are estimated based on a least-squares optimization. Averaging the transformed intensities gives the consensus signals, which are independent of the voltage settings of the photomultiplier tube. 6) Raw data was retrieved with GenePix Pro 6.0 software from Axon Instruments. 7) Data processing was performed. 8) The scans were performed with a resolution of 10 um.
Description
This record represents all technological and biological replicates (i.e. from the same and different biological source, respectively) that were performed with this particular sediment extract at this particular concentration. The Dye-Swap Design was utilized. Ehingen_extract_1_zp8-32.high_2.gpr: test=Cy5, reference=Cy3 Ehingen_extract_1_zp8-32.low.gpr: test=Cy5, reference=Cy3 Ehingen_extract_1_zp8-32.medium.gpr: test=Cy5, reference=Cy3 Ehingen_extract_1_zp8-87.high_2.gpr: test=Cy3, reference=Cy5 Ehingen_extract_1_zp8-87.low.gpr: test=Cy3, reference=Cy5 Ehingen_extract_1_zp8-87.medium.gpr: test=Cy3, reference=Cy5 Ehingen_extract_2_zp8-18.high_4.gpr: test=Cy5, reference=Cy3 Ehingen_extract_2_zp8-18.low.gpr: test=Cy5, reference=Cy3 Ehingen_extract_2_zp8-18.medium.gpr: test=Cy5, reference=Cy3 Ehingen_extract_2_zp8-28.high_2.gpr: test=Cy3, reference=Cy5 Ehingen_extract_2_zp8-28.low.gpr: test=Cy3, reference=Cy5 Ehingen_extract_2_zp8-28.medium_2.gpr: test=Cy3, reference=Cy5 Ehingen_extract_3_zp8-29.high.gpr: test=Cy5, reference=Cy3 Ehingen_extract_3_zp8-29.low.gpr: test=Cy5, reference=Cy3 Ehingen_extract_3_zp8-29.medium.gpr: test=Cy5, reference=Cy3 Ehingen_extract_3_zp8-36.high_2.gpr: test=Cy3, reference=Cy5 Ehingen_extract_3_zp8-36.low.gpr: test=Cy3, reference=Cy5 Ehingen_extract_3_zp8-36.medium.gpr: test=Cy3, reference=Cy5
Data processing
1) Raw data are retrieved with GenePix. 2) Quality control on spot level. 3) Local background correction. 4) Averaging of the two spots for each gene. 5) Consensus signals are calculated from low, medium and high scan based on a Least Squares Algorithm. 6) Log transformation. 7) Locally weighted regression smoother (LOESS). 8) Scaling to a common MAD. 9) t-test. 10) Adjustment of p-values for multiplicity. 11) Quality control on array level.
