Magmax express 96 system

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).

Total RNA from cells was extracted using the automated MagMax Express 96 system (Applied Biosystems) using the Magmax-96 total RNA isolation kit (Life Technologies). Reverse transcription (RT) was performed as previously reported [48 (link)]. Real-time polymerase chain reactions (PCRs) were performed with iQ SYBR Green supermix (Bio-Rad) in a CFX96 system from Bio-Rad, using specific primers for each gene (Table 1). The amount of each transcript was expressed by the formula: 2^{ct(β-actin or CD3)−ct(gene)}, ct being the point at which the fluorescence rises appreciably above the background levels.