Data from: Counting cytoplasmic incompatibility factor mRNA using digital droplet PCR

Wolbachia 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. Wolbachia's cifB gene triggers CI in
males, while cifA, expressed in females, rescues embryos from CI-induced
lethality. In some systems, cifA also contributes to CI induction. CI
strength—the percentage of embryos that die from CI—is a key determinant
of Wolbachia's prevalence in host populations, and cifB mRNA levels
in testes generally correlate with CI strength. Yet, cifB'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 cifA and cifB mRNA from wMel Wolbachia in Drosophila
melanogaster. These assays count cif transcripts alongside a synthetic
spike-in RNA or a D. melanogaster housekeeping gene to normalize for
technical or biological variation. These assays have a limit of detection
of about 1 cifA and 3 cifB copies per reaction. We expect these methods to
be useful for mosquito-control programs that use wMel to block the spread
of pathogens from Aedes aegypti to humans. Moreover, the oligos were
designed with homology to cifA and cifB sequences from at least 33
Wolbachia strains, suggesting utility beyond wMel. These methods will
allow researchers to measure cif mRNA levels from individual insect
tissues, enabling efforts to pair molecular and phenotypic data at
unprecedented resolutions.