Workflow to extract and process RNA from Drosophila testes with and without the endosymbiontWolbachia v1

This protocol describes a complete workflow for extracting and processing RNA from Drosophila testes, including those harboring the endosymbiont Wolbachia. We begin by extracting and purifying RNA using TRIzol:chloroform phase separation, followed by a TURBO DNase Kit treatment to remove contaminating genomic DNA. cDNA is then synthesized using the SuperScript IV VILO Master Mix. Before DNase treatment, this method consistently yields approximately 52.5 ng of RNA per testes pair, with A260/A280 of ~1.94 and A260/A230 of ~1.45. Using digital droplet PCR (ddPCR) to quantify the Wolbachia cifA gene and the Drosophila β-spectrin gene, we confirmed that the DNase treatment completely eliminates genomic DNA contamination. This workflow is suitable for performing sensitive, accurate, and precise RT-ddPCR assays to measure both Wolbachia and Drosophila mRNA levels.