Use of Genomic DNA as an Indirect Reference for Identifying Gender-Associated Transcripts in Morphologically Identical, but Chromosomally Distinct, Schistosoma mansoni Cercariae

Background: The use of DNA microarray technology to study global Schistosoma gene expression has led to the rapididentification of novel biological processes, pathways or associations. Implementation of standardized DNA microarrayprotocols across laboratories would assist maximal interpretation of generated datasets and extend productive applicationof this technology.

Methodology/Principal Findings: Utilizing a new Schistosoma mansoni oligonucleotide DNA microarray composed of 37,632elements, we show that schistosome genomic DNA (gDNA) hybridizes with less variation compared to complex mixed pools ofS. mansoni cDNA material (R = 0.993 for gDNA compared to R = 0.956 for cDNA during ‘self versus self’ hybridizations).Furthermore, these effects are species-specific, with S. japonicum or Mus musculus gDNA failing to bind significantly to S.mansoni oligonucleotide DNA microarrays (e.g R = 0.350 when S. mansoni gDNA is co-hybridized with S. japonicum gDNA).Increased median fluorescent intensities (209.9) were also observed for DNA microarray elements hybridized with S. mansonigDNA compared to complex mixed pools of S. mansoni cDNA (112.2). Exploiting these valuable characteristics, S. mansonigDNA was used in two-channel DNA microarray hybridization experiments as a common reference for indirect identification ofgender-associated transcripts in cercariae, a schistosome life-stage in which there is no overt sexual dimorphism. This led tothe identification of 2,648 gender-associated transcripts. When compared to the 780 gender-associated transcripts identifiedby hybridization experiments utilizing a two-channel direct method (co-hybridization of male and female cercariae cDNA),indirect methods using gDNA were far superior in identifying greater quantities of differentially expressed transcripts.Interestingly, both methods identified a concordant subset of 188 male-associated and 156 female-associated cercarialtranscripts, respectively. Gene ontology classification of these differentially expressed transcripts revealed a greater diversity ofcategories in male cercariae. Quantitative real-time PCR analysis confirmed the DNA microarray results and supported thereliability of this platform for identifying gender-associated transcripts.

Conclusions/Significance: Schistosome gDNA displays characteristics highly suitable for the comparison of two-channelDNA microarray results obtained from experiments conducted independently across laboratories. The schistosometranscripts identified here demonstrate, for the first time, that gender-associated patterns of expression are already wellestablished in the morphologically identical, but chromosomally distinct, cercariae stage.