Validation of the RNA isolation method from single zebrafish embryos. (a) RNA RIN values and (b) yields from: Single, individual embryos (4); Semi-single, homogenized, pooled, and split embryo material (4, see text); Developmental, embryos from the 16-cell to 8-somite stage (8); Unfertilized, unfertilized eggs (30); Epibolic, embryos from dome stage to 90% epiboly (186). Stages and RNA yield could not be linked. Note that RIN values show overlap because of a single decimal place measurement accuracy. (c) Mean, unnormalized log2 signal intensities from microarray analysis (smooth bar, foreground signal and scatter board bar, background signal). (d) Principal component analysis (PCA) of the unnormalized log2 ratios (test/reference) from the Single and Semi-single samples. (e) Spearman correlations showing the similarity of the unnormalized microarray data from Single and Semi-single samples.