All exposures to the ORCs were conducted on adult female daphnids 14-16 days old and occurred at 23.50 C for 24 hours
Growth protocol
Genetically homogeneous Daphnia magna, purchased from Aquatic Research Organisms (Hampton, NH), were cultured in MHRW and maintained at 23.50 C according to standard protocols (Weber, 1993; Lewis et al., 1994). Weber, C. I. (1993) Methods for Measuring the Acute Toxicity of Effluent and Receiving Waters to Freshwater and Marine Organisms. U.S. Environmental Protection Agency: EPA/600/4-90/027F; Lewis, P. A., Klemm, D. J., Lazorchak, J. M., Norberg-King, T. J., Peltier, W. H. & Heber, M. A. (1994) Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. US Environmental Protection Agency: EPA/600/4-91/002
Extracted molecule
total RNA
Extraction protocol
Whole daphnids (40 pooled for each exposure) were initally preserved in RNAlater (Ambion). They were later transferred to liquid nitrogen and ground with a morter and pestle. RNA was isolated using Trizol according to standard methods (Invitrogen, Carlsbad, CA).
Label
cy3
Label protocol
cDNA was synthesized using Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) from total RNA in the presence of aminoallyl labeled dUTP. Fluorescence labeling proceeded by incubating the aminoallyl labeled cDNA with Cy5 or Cy3 fluorescent dyes (Amersham Biosciences, Piscataway, NJ). The dyes were switched for each technical replicate so that the control cDNA was labeled with Cy3 in one hybridization and Cy5 in the other.
All exposures to the ORCs were conducted on adult female daphnids 14-16 days old and occurred at 23.50 C for 24 hours
Growth protocol
Genetically homogeneous Daphnia magna, purchased from Aquatic Research Organisms (Hampton, NH), were cultured in MHRW and maintained at 23.50 C according to standard protocols (Weber, 1993; Lewis et al., 1994). Weber, C. I. (1993) Methods for Measuring the Acute Toxicity of Effluent and Receiving Waters to Freshwater and Marine Organisms. U.S. Environmental Protection Agency: EPA/600/4-90/027F; Lewis, P. A., Klemm, D. J., Lazorchak, J. M., Norberg-King, T. J., Peltier, W. H. & Heber, M. A. (1994) Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. US Environmental Protection Agency: EPA/600/4-91/002
Extracted molecule
total RNA
Extraction protocol
Whole daphnids (40 pooled for each exposure) were initally preserved in RNAlater (Ambion). They were later transferred to liquid nitrogen and ground with a morter and pestle. RNA was isolated using Trizol according to standard methods (Invitrogen, Carlsbad, CA).
Label
cy5
Label protocol
cDNA was synthesized using Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) from total RNA in the presence of aminoallyl labeled dUTP. Fluorescence labeling proceeded by incubating the aminoallyl labeled cDNA with Cy5 or Cy3 fluorescent dyes (Amersham Biosciences, Piscataway, NJ). The dyes were switched for each technical replicate so that the control cDNA was labeled with Cy3 in one hybridization and Cy5 in the other.
Hybridization protocol
The labeled cDNA pools from the unexposed and exposed D. magna were mixed and hybridized to the Daphnia microarray (protocols available at http://cmgm.stanford.edu/pbrown/mguuide).
Scan protocol
Scanning and quantification was performed using an arrayWoRx Biochip Reader (Applied Precision, Issaquah, WA) and GenePix software version 3.01 (Axon Instruments, Union City, CA).
Description
1.194 mg/L TNT
Data processing
Technical replicates were normalized to remove possible non-linearity, if any, and checked for homogeneity using boxplots. As an alternative to between-slide normalization we applied an approach based on sequential single-slide data analysis and utilized the α-outlier-generating model and outlier regions approach to identify differentially expressed cDNAs. Details of the method can be found in: Loguinov, A. V., Mian, I. S. & Vulpe, C. D. (2004) Exploratory differential gene expression analysis in microarray experiments with no or limited replication. Genome Biol. 5, R18.
