Direct zol rna 96 magbead zymo kit

Upon completion of each KD experiment, individual whole mosquitoes were anesthetized by chilling and placed in 1.5-ml microfuge tubes (Sarstedt, Nümbrecht, Germany) containing 300 μl of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and a 2.8-mm ceramic bead. Samples were homogenized on a Bead Ruptor Elite (Omni International, USA) and then frozen at −80°C. Total RNA was extracted with the Direct-zol RNA 96 Magbead Zymo kit (Zymo Research, Irvine, CA) according to the manufacturer’s protocol. Following this step, the samples were processed using an automatic magnetic bead purification system (MagMAX Express 96 system, Applied Biosystems). RNA was eluted in 50 μl RNase free water. RNA was then treated with 5 units of DNase I (Sigma-Aldrich) at room temperature for 15 min, followed by inactivation with 50 mM EDTA at 70°C for 10 min. To measure Wolbachia loads, extractions for both RNA and DNA were performed using the column-based Direct-zol DNA/RNA Miniprep kit. RNA was eluted in 50 μl RNase free water, followed by DNA elution in 50 μl of Direct-zol DNA Elution Buffer. Total RNA and DNA concentrations were determined with a NanoDrop model 2000/2000C (Thermo Scientific, Waltham, MA).

Individual tissues from single mosquitoes were placed in 300 μl of TriReagent (Sigma‐Aldrich), or 200 μl for salivary glands, and homogenized on a Bead Ruptor Elite (Omni International, USA) using a 2.8 mm ceramic bead. Total RNA was extracted with the Direct‐zol RNA 96 Magbead Zymo kit (Zymo Research) according to the manufacture's protocol on a MagMAX Express 96 system (Applied biosystems), and RNA was eluted in 50 μl RNase free water. RNA was treated with 5 units of DNase I (Sigma‐aldrich) at room temperature (RT) for 15 min, followed by inactivation with 50 mM EDTA at 70°C for 10 min. To measure Wolbachia DNA, RNA and DNA combination extractions were performed using the column‐based Direct‐zol DNA/RNA Miniprep kit. RNA was eluted in 50 μl RNase free water, followed by DNA elution in 50 μl of Direct‐zol DNA Elution Buffer, and RNA was treated with DNase I (as above).