RNA Extraction

RNA was extracted from male and female mites separately using the Trizol extraction method (Chomczynski and Mackey, 1995) with some modifications to the original protocol. Briefly, mites were collected individually and then male and female mites were pooled in separate tubes. Mites were homogenized in 500 µl Trizol using a plastic pestle. After homogenization, samples were mixed for 10 min at room temperature in a shaker followed by centrifugation at 15000 x g for 10 min at 4°C. The supernatant was transferred to a new tube and incubated for 5 min at room temperature to permit complete dissociation of the nucleoproteins. One hundred µl chilled chloroform was added to the samples, mixed, and incubated for 10 min at 4°C. Samples were centrifuged at 15000 x g for 15 mins to obtain an aqueous phase. The aqueous phase was transferred to a new tube and then 600 µl of isopropanol was added before storage overnight at -20°C. The following day, samples were centrifuged for 15 min at 4°C followed by washing the pellets in 70% ethanol. RNA pellets were dried, and RNA samples were resuspended in sterile water and quantified using a NanoDrop instrument (Thermo Fisher Scientific, Waltham, MA).