Differential expression analysis of genes relevant to the moult cycle of Portunus pelagicus

A holistic approach to examine differential gene expression profiles of transcripts relevant to the moulting process, across all moult cycle stages, was used in this study. Custom cDNA microarrays were constructed for P. pelagicus. The printed arrays contained 5000 transcripts derived from both the whole organism, and from individual organs such as the brain, eyestalk, mandibular organ and Y-organ from all moult cycle stages. A total of 556 clones were sequenced from the cDNA libraries used to construct the arrays. These cDNAs represented 217 singlets and 62 contigs, resulting in 279 unique putative genes. Moult cycle-related differential expression patterns were observed for many transcripts. Keywords: cyclic moult stage comparison of the crab Portunus pelagicus In order to identify differential gene expression across moult stages, two consecutive moult stages were compared on each array in a dual colour (Cy3 and Cy5) experiment. RNA samples were pooled across subjects in order to reduce the effect of biological variation (nine subjects were pooled across a total of three arrays) for each moult stage comparison. Equal amounts of total RNA from three crabs in one moult stage, were pooled, and compared against equal amounts of total RNA pooled from three crabs in another moult stage, on one array. This was repeated three times in total, the different moult stages were labelled with Cy3 or Cy5 respectively. Consecutive moult stages were compared in the following format; postmoult (Cy3) with intermoult (Cy5), intermoult (Cy3) with early premoult (Cy5), early premoult (Cy3) with late premoult (Cy5), late premoult (Cy3) with ecdysis (Cy5), and ecdysis (Cy3) with postmoult (Cy5).