Figure 5

BNF-ANF treatment exposures and microarray loop design: each sample is hybridized to 2 arrays using both Cy3 and Cy5 labeled fluorophores. The loop consisted of Cy3 and Cy5 labeled embryo aRNAs from 4 biological samples and six different treatments (T1-T6: control, 1 μg/L BNF, 50 μg/L ANF, 100 μg/L ANF, 1 μg/L BNF + 50 μg/L ANF, 1 μg/L BNF + 100 μg/L ANF). Anticipated treatment effects on AHR and CYP450IA genes are shown with up and down green/red arrows above the treatments. 48 biological samples were hybridized to 24 microarrays. Each array had different combinations of biological samples, so that the most direct comparisons (i.e., 50 μg/L ANF resistant embryo and 50 μg/L reference embryo) are hybridized to the same array. The loop formed was T1S → T1R → T2S → T2R → T3S → T3R → T4S → T4R → T5S → T5R → T6S → T6R → T1S → T2S → T3S → T4S → T5S → T6S → T1S → T1R → T2R → T3R → T4R → T5R → T6R, where each arrow represents a separate hybridization (array) with the biological sample at the base of the arrow labeled with Cy3 and the biological pool at the head of the arrow labeled with Cy5. T1-6 is treatment, and S and R represent reference and resistant embryos, respectively. A shorter example of the loop design shows treatments as numbers, and arrows as separate hybridization (array).