Polypropylene pestles

Female mosquitoes maintained on the two different diets were collected at different times after adult eclosion (2, 3, 4, 6 and 7 days) and immobilized by exposure to ice. Ovaries were dissected by performing an incision in the thorax, cutting the last abdominal segment and pulling out the ovaries^{28 (link)}. Triplicates samples of 10 ovaries each were placed in 1.5 mL Eppendorf tubes, and 10 µL of a mix of labeled internal standard were added (EquiSplash Lipidomix, Avanti Polar Lipids, Alabaster, AL). After adding 100 µL of butanol/methanol and 3 µL of butylated hydroxytoluene, samples were homogenized for 10 s using polypropylene pestles (Fisher Scientific, Pittsburgh, PA), and a handheld cordless motor. The pestles were then rinsed with 200 µL of butanol/methanol. All tubes were sonicated for 30 min, and then centrifuged for 10 min. The supernatant was transferred into autosampler vials with 300 µL silanized glass inserts (Thermo Fisher Scientific, Waltham, MA).

Frozen diaphragms were cut and weighed, yielding 10–20 mg of tissue. Samples underwent three freeze–thaw cycles using liquid nitrogen, then homogenized in Trizol Reagent (Ambion, Waltham, MA, USA)) using polypropylene pestles (Fisher Scientific, Hampton, NH, USA) and an Eppendorf tube. RNA purification was performed using a Direct-zol^TM RNA miniprep kit (Zymo, Irvine, CA, USA) according to the manufacturer’s instructions. Post-purification was performed through an in-solution Dnase I treatment using <10 µg of RNA sample, Dnase I (Zymo, Irvine, CA, USA), DNA digestion buffer (Zymo, Irvine, CA, USA), and water and incubated at room temperature for 15 min. Three volumes of Trizol to one volume of the treated sample were added. The RNA purification step using the Direct-zol^TM RNA miniprep kit was then repeated, and the RNA was suspended in 40 µL of RNAase-free water. RNA concentration and quality were quantified using a DeNovix DS-11 spectrophotometer. All samples were verified to have a 260/280 ratio above 1.9 and a 260/230 ratio between 2.0–2.2.
cDNA synthesis was performed using 1 µg of total RNA with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) following the manufacturer’s instructions.