whole body mRNA Strains: 200-800 zebrafish from AB*,AB2O2,AB2O2 Golden and AB2O2 Leo whole body mRNA Strains: 200-800 zebrafish from AB*,AB2O2,AB2O2 Golden and AB2O2 Leo
Biomaterial provider
Fish Facility at the Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe
Treatment protocol
Acrylamide (CH2=CHCONH2), Aroclor 1254 (PCB), Arsenic(III) Oxide (As2O3), tBHQ (tert-Butylhydroquinone (CH3)3CC6H3-1,4-(OH)2), Cadmiumchloride (CdCl2.2H2O), 4-Chloroaniline (ClC6H4NH2), DDT (1,1-Bis-(4-Chlorphenyl)-2,2,2-Trichlorethan (ClC6H4)2CHCCl3), Lead (II)chloride (PbCl2), Methylmercurychloride (CH3ClHg), TCDD (2,3,7,8-Tetrachlorodibenzo-p-dioxin) and Valproic acid (CH3(CH2)4CO2H) were purchased from Sigma-Aldrich. Zebrafish wild type strains AB, ABO and Tuebingen were kept and bred. Embryos were grown in embryo medium (60 ug/ml Instant Ocean, Red Sea Fish Pharm Ltd.) Different numbers of embryos were exposed to the chemicals: between 4 to 24 hpf (600 embryos), 24 to 48 hpf (400 embryos) and 96 to 120 hpf (200 embryos). Embryo medium alone (Cd, Hg, Pb, As, VA, AA) or 0.2 % ethanol in embryo medium (tBHQ , 4CA, PCB, DDT) or 0.5% DMSO, 28mg/l toluene in embryo medium (TCDD) were used as vehicle controls. The toxin concentrations were adjusted in such a way that embryo death was minimal. The few dead embryos were discarded before preparation of RNA.
Growth protocol
as described in Westerfield 1993
Extracted molecule
polyA 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
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 that were not exposed to a toxic environment
Strains: 200-800 zebrafish from AB*,AB2O2,AB2O2 Golden and AB2O2 Leo whole body mRNA
Biomaterial provider
Fish Facility at the Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe
Treatment protocol
Acrylamide (CH2=CHCONH2), Aroclor 1254 (PCB), Arsenic(III) Oxide (As2O3), tBHQ (tert-Butylhydroquinone (CH3)3CC6H3-1,4-(OH)2), Cadmiumchloride (CdCl2.2H2O), 4-Chloroaniline (ClC6H4NH2), DDT (1,1-Bis-(4-Chlorphenyl)-2,2,2-Trichlorethan (ClC6H4)2CHCCl3), Lead (II)chloride (PbCl2), Methylmercurychloride (CH3ClHg), TCDD (2,3,7,8-Tetrachlorodibenzo-p-dioxin) and Valproic acid (CH3(CH2)4CO2H) were purchased from Sigma-Aldrich. Zebrafish wild type strains AB, ABO and Tuebingen were kept and bred. Embryos were grown in embryo medium (60 ug/ml Instant Ocean, Red Sea Fish Pharm Ltd.) Different numbers of embryos were exposed to the chemicals: between 4 to 24 hpf (600 embryos), 24 to 48 hpf (400 embryos) and 96 to 120 hpf (200 embryos). Embryo medium alone (Cd, Hg, Pb, As, VA, AA) or 0.2 % ethanol in embryo medium (tBHQ , 4CA, PCB, DDT) or 0.5% DMSO, 28mg/l toluene in embryo medium (TCDD) were used as vehicle controls. The toxin concentrations were adjusted in such a way that embryo death was minimal. The few dead embryos were discarded before preparation of RNA.
Growth protocol
as described in Westerfield 1993
Extracted molecule
polyA 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
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 an 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. 6) Raw data was retrieved with GenePix Pro 6.0 software from Axon Instruments . 7) Data processing was performed 8) The absolute intensity values span the range from 0 to 65536. 9) The scans were performed with a resolution of 10 um.
Description
This file contains all technological and biological replicates (i.e. from the same and different biological source, repspectively) that were performed with this particular toxin at this particular concentration. The Dye-Swap Designe was utilised.
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.
