An important aspect of our results is that we do not observe the complete breakdown of this spermatogenesis regulatory pathway in testes of sterile hybrids. Instead, multiple transcripts are expressed at levels within the range of that observed within species. Relatedly, in sterile F1 hybrids of these species (and various introgression lines), spermatogenesis appears to proceed to and through meiosis and arrest thereafter [26,27], but this conclusion relies on microscopic observations that may have missed subtle defects. If disruptions in particular segments of this transcriptional network (Figure 1) are associated with hybrid sterility, it may be possible to use a directed ‘‘candidate gene’’ approach to identify specific failed interactions. If we can extrapolate from the results presented here, a logical place to look for such failed interactions would be with interactions involving late-acting meiotic-arrest genes of the cannonball (can) class [28]. With help from Applied Biosystems, we have developed a protocol for consistent real-time PCR quantification of transcript abundance from the testes derived from single Drosophila. While expression analyses from even single cells have been possible for some time [e.g., 29], the preparations sometimes rely upon the use of cultured cells, polyadenylated transcripts, RNA amplification steps [e.g., 30], or other manipulations or limitations. Prior to using this approach, we attempted RNA isolation and amplifica- tion using a commercially available kit, but the amplified RNA degraded after a few freeze-thaws. Other more direct RNA isolation approaches we attempted did not have high repeatability across technical replicates or had issues including false amplifica- tion or contamination. The approach utilized here involves RNA isolation from testes followed by cDNA synthesis for use as a template in RQ-PCR. 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