Using reverse transcription digital droplet PCR to count rare cytoplasmic incompatibility factor transcripts v1

This protocol details four reverse transcription digital droplet polymerase chain reaction (RT-ddPCR) assays to count cifA and cifB mRNA from wMel Wolbachia in Drosophila melanogaster. These assays count cif transcripts in duplex with either a synthetic spike-in RNA to normalize for technical variation or a D. melanogaster housekeeping gene to normalize for biological variation. With a limit of detection of about 1 cifA and 3 cifB copies per 20 μL reaction, the assays provide accurate and precise measurements across a wide dynamic range. We designed oligos 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 within individual insect tissues, enabling efforts to pair molecular and phenotypic data at unprecedented resolutions.