Figure 1. Drosophila spermatogenesis regulatory pathway [adapted from 17,18]. doi:10.1371/journal.pone.0003009.g001 Here, we present a novel protocol for consistent real-time PCR quantification of transcript abundance from the testes of a single Drosophila. We use this approach on four spermatogenesis-related transcripts to address two questions relating to hybrid misexpres- sion in Drosophila simulans clade species, which diverged from each other approximately 250,000 years ago [21]. First, we test whether the same hybrid misexpression in whole bodies is also apparent when testes are examined alone. Second, we test the hypothesis of Moehring et al. [13] that hybrid misexpression is preferentially localized to late-stage downstream loci relative to early-stage loci of known spermatogenetic pathways in this system. If this hypothesis is confirmed, that would further support, albeit not prove, the hypothesis that gene expression disruptions could contribute to hybrid sterility in this system. Drosophila Hybrid Disruptions Materials and Methods Drosophila Strains Drosophila simulans Florida City strain and D. mauritiana Synthetic strain were maintained on standard sugar-yeast-agar medium on a 12-hour light-dark cycle at 20uC. Virgin D. simulans females were crossed with D. mauritiana males. F1 males were collected, housed three days post-eclosion, and nucleic acids were extracted immediately thereafter from either testes or whole fly body. Development of Custom TaqMan Gene Expression Assays The D. melanogaster DNA sequences of four genes known to be involved in spermatogenesis (always early (aly), cookie monster (comr), don juan (dj), and Mst84D) and an endogenous control gene (Actin5C), were collected from NCBI (http://www.ncbi.nlm.nih. gov/). The D. melanogaster DNA sequence was BLASTed against the D. simulans genome using Drosophila Species Genomes BLAST (http://insects.eugenes.org/species/blast/). The D. simu- lans genes were aligned with the orthologous D. melanogaster sequences. Primers were designed to sequence regions of the genes with minimal nucleotide differences between the two species. Sequencing was performed for our D. simulans Florida City and D. mauritiana Synthetic strains. The resulting sequence reads were aligned to reveal polymorphisms between the orthologs. TaqMan probe and primer sets were designed to avoid polymorphisms, while maximizing the efficiency of the amplification. When possible, probes were designed to cross an intron/exon boundary. Probes recognizing the four genes of interest were labeled with a 59 FAM reporter fluorophore and the endogenous control gene was labeled with a 59 VIC fluorophore. Probes and primers were synthesized by Applied Biosystems (Foster City, CA). The probe and primer sequences are listed in Table S1. RNA Extraction and cDNA Synthesis Whole-fly RNA extractions were prepared according to a previously published protocol [22], followed by cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems. Testes were carefully dissected from anesthetized flies in insect Ringer’s solution and immediately processed using the TaqMan Gene Expression Cells-to-CT Kit from Applied Biosystems with a modified protocol (see Methods S1 online for the detailed protocol). We employed both technical and biological replication (3–4 separate testes dissections), with the latter involving independent reverse transcription reactions and subsequent RQ- PCRs from testes of each fly dissected. We also performed both no-template controls and reactions with RNA that was not reverse transcribed (to confirm the absence of genomic DNA contamina- tion). Finally, we did also amplify and sequence both parental strains and F1 hybrids to confirm that the F1 hybrid samples were heterozygous at all polymorphisms identified (and not contami- nants). Data Analyses Real-time quantitative PCR was performed on an ABI 7000 PRISM Sequence Detection System. Raw CT values were collected and analysis was performed according to the 22DDCT method [23], using Actin5C to normalize estimates of relative expression. The same general trends were apparent in the raw CT values for the focal genes as in the normalized data, suggesting Actin5C normalization did not skew the results. Whole body expression data for always early were taken directly from Noor [20]. For visualization in Table 1, relative expression was further PLoS ONE | www.plosone.org 2 August 2008 | Volume 3 | Issue 8 | e3009