Pollution Effects on Metabolic Gene Expression During Late Organodifferenatiation of Sensitive, Resistant and Hybrid Vertebrate Embryos

The goal was to determine differences in gene expression of 384 metabolic genes during late organogenesis among embryos of parents from a clean site (Magotha Bay, VA) and polluted site (Elizabeth RIver, VA). Hybrid gene expression was examined to explore parental effect. Embryo morphology and heart rate during late organogenesis and pre-hatching was assessed in order to determine phenotypic differences between embryo groups. Labeled RNA samples were hybridized to slides according to the loop design, a randomized incomplete block design that involves constraints of different treatment types so that every comparison is represented by a direct contrast on a microarray. Each sample was hybridized to 2 arrays using both Cy3 and Cy5 flourescent dyes, which is a simple and effective way for the direct comparison of two samples, and for reducing technical variation (Churchill, 2002). The loop consisted of Cy3 and Cy5 labeled individual embryo aRNA, resulting in total of 40 replicates (20 biological replicates x 2 dyes for each. Since each array consisted of 4 replicates of metabolic genes, there were 8 total number of gene expression measurement for each embryo (embryo aRNA x 2 dyes x 4 gene replicates = 8 measurements / embryo). The loop formed was CC5 -- PP41 --CP21--PC26--CC9--PP32--CP13--PC24--CC6_PP34--CP16--PC28--CC10--PP33-CP18-PC27-CC7-PP40-CP17-PC25-CC5, where each dash represents a separate hybridization (array) with the individual biological sample at the left of the dash labeled with Cy3 and the biological pool at the right of the dash labeled with Cy5. C denotes clean site parent, P denotes polluted site parent; Hence, the CC, CP, PC, and PP represent treatments (CC = embyo of a mother fomr the celan site and the fater from the clean site; CP = embryo of a mother from the clean site and fatehr from polluted site, etc...). A nuber after the treament (CC5, so #5) represents the individual embryo utilized (5th embryo in CC treamtent).