Counting cytoplasmic incompatibility factor mRNA using digital droplet PCR.

bacteria inhabit over half of all insect species and often spread through host populations via efficient maternal transmission and cytoplasmic incompatibility (CI), killing aposymbiotic embryos when fertilized by symbiotic males. 's gene triggers CI in males, while , expressed in females, rescues embryos from CI-induced lethality. In some systems, also contributes to CI induction. CI strength-the percentage of embryos that die from CI-is a key determinant of 's prevalence in host populations, and mRNA levels in testes generally correlate with CI strength. Yet, 's rarity can hamper precise quantification, necessitating tissue pooling for reverse transcription quantitative PCR (RT-qPCR) to achieve reliable measurements, obscuring variation at the level of individual insect tissues. Here, we present four RT digital droplet PCR (RT-ddPCR) assays to count rare and mRNA from Mel in . These assays count transcripts alongside a synthetic spike-in RNA or a housekeeping gene to normalize for technical or biological variation. These assays have a limit of detection of about 1 and 3 copies per reaction. We expect these methods to be useful for mosquito-control programs that use Mel to block the spread of pathogens from to humans. Moreover, the oligos were designed with homology to and sequences from at least 33 strains, suggesting utility beyond Mel. These methods will allow researchers to measure mRNA levels from individual insect tissues, enabling efforts to pair molecular and phenotypic data at unprecedented resolutions.