Figure 1

Experimental design. A total of 20 microarray hybridizations were performed, addressing biological variation through the use of 12 biologically independent samples for each sex, and technical variation through Cy3/Cy5 dye reversal as follows. (A) For each of the four embryonic stages (ed4, 6, 8 and 10), two samples per sex were generated by pooling equal amounts of total RNA from four individual male (M) or female (F) whole heads (ed4 and 6) or brains (ed8 and 10). Each RNA pool was converted to two cDNA samples, tagged with Cy3 and Cy5-capture sequence (Genisphere), respectively, and hybridized to two microarray slides, as illustrated by arrows. Arrowhead indicates Cy5-labeled sample. (B) For ed10, total RNA from four individual male (M) and female (F) brains were separately tagged with either Cy3 or Cy5-capture sequence during cDNA synthesis, and analyzed on four microarray hybridizations with dye-reversal as illustrated by arrows.