population: New Bedford Harbor tissue: Whole embryo treatment: PCB-126 days post-fertilization: 15
Treatment protocol
After non-fertile eggs were culled, embryos were exposed to vehicle (DMSO; 0.1%) or PCB-126 (50 nM) in filtered seawater (salinity 25 part per thousand, 65 embros per 20 ml in glass petri dish) for 4 hr at 20º C. After exposure, the embryos were washed in filtered seawater and incubated at 20º C under a 14-h light, 10-h dark cycle. At 5-, 10-, and 15-dpf, embryos were collected as three pools of 20 embryos from each treatment group and flash frozen in liquid nitrogen and stored at -80º C until used for RNA isolation
Growth protocol
F. heteroclitus adults were collected from New Bedford Harbor, MA USA and Scorton Creek, MA USA in May and June of 2007. For each site, eggs from 8 females (~1100 total) were fertilized using minced testes from 5 males. After non-fertile eggs were culled, embryos were exposed to vehicle (DMSO; 0.1%) or PCB-126 (50 nM) in filtered seawater (salinity 25 part per thousand, 65 embros per 20 ml in glass petri dish) for 4 hr at 20º C. After exposure, the embryos were washed in filtered seawater and incubated at 20º C under a 14-h light, 10-h dark cycle. At 5-, 10-, and 15-dpf, embryos were collected as three pools of 20 embryos from each treatment group and flash frozen in liquid nitrogen and stored at -80º C until used for RNA isolation
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from pools of embryos (20 embryos per pool) using STAT-60 (Tel-Test).
Label
Cy5
Label protocol
RNA for hybridization was prepared by one round of amplification (aRNA) using the Amino Allyl MessageAmp aRNA Kit (Ambion, Austin, TX, USA) to form copy template RNA by T7 amplification. Amino-allyl UTP was incorporated into targets during T7 transcription, and resulting amino-allyl aRNA was coupled to Cy3 and Cy5 dyes (GE Healthcare, Piscataway, NJ, USA).
Hybridization protocol
Labeled aRNA samples (2 pmol dye/ul) were hybridized to slides in 10 ul of hybridization buffer [50% formamide buffer, 5x SSPE, 1% sodium dodecyl sulfate, 0.2 mg/ml bovine serum albumin, 1 mg/ml denatured salmon sperm DNA (Sigma), and 1 mg/ml RNAse free poly(A) RNA (Sigma)] for 44 hours at 42º C.
Scan protocol
Arrays were scanned using a ScanArray Express 4000 (Perkin Elmer). Resulting 16 bit Tiff Images were quantified using ImaGene® (Biodiscovery, Inc.) spotfinding software.
Data processing
Log2 measures of gene expression were normalized using a linear mixed model in JMP Genomics 3.2 (SAS, Cary, NC, USA) to remove the effects of dye (fixed effect) and array (random effect) following a joint regional and spatial Lowess transformation in MAANOVA version 0.98.8 for R to account for both intensity and spatial bias. The model was of the form yij = μ + Ai + Dj + (AxD)ij + εij where, yij is the signal from the ith array with dye j, μ is the sample mean, Ai and Dj are the overall variation in arrays (arrays 1-80) and dyes (Cy3 and Cy5), (AxD)ij is the array x dye interaction and εij is the stochastic error. Because we were not interested in differences due to developmental stage, we analyzed residuals from the above model separately by time (e.g., separately for times 5, 10 and 15 dpf). Thus, residuals from this model were used for gene-by-gene analyses by time of population and treatment effects, using population, treatment and dye as fixed effects, and array as a random effect. The model was rijn = μ + Ai + Dj + Pn + Tk + PnxTk + εijn where Pn is the nth population and Tk is the kth treatment (DMSO or PCB-126). For all mixed model analyses, we used a false discovery rate (FDR) p-value of < 0.01 to control for multiple testing
Transcriptomic assessment of resistance to effects of an aryl hydrocarbon receptor (AHR) agonist in embryos of Atlantic killifish (Fundulus heteroclitus) from a marine Superfund site
Data table header descriptions
ID_REF
VALUE
Values represent least square mean values from the mixed model normalization of log2, loess transformed raw data.
