Ckmix 2 ml

In order to test, whether the DREAM method is suitable for the deorphanization of broadly as well as narrowly tuned ORs and different chemosensory receptor types transcription changes of receptor genes were measured after flies were exposed to high odorant concentrations. Three hours after the beginning of the light phase the odorants or pure solvents were introduced into the rearing vials. To avoid interaction of the chemicals with the artificial diet, 30 μL of the odorants or solvents, respectively, were applied into the well of a detached 2.0 mL reaction tube lid. After an exposure time of 5 h, flies were transferred into new, empty vials, and cooled down for 5 min in a −80°C freezer. The flies were then maintained at −20°C until dissection. For each biological replicate, 50 manually removed fly heads (male–female ratio 1:1) were collected in 2.0-mL microcentrifuge tubes containing mixed zirconium oxide beats of 1.4 and 2.8 mm (CKmix-2 mL, Bertin Instruments) as well as 600 μL TRIzol^® (Sigma-Aldrich). For the sample collection of the pheromone treatments and corresponding controls, only the heads of male flies were used. During dissection, samples were stored on ice. After dissection samples were homogenized in a bead mill (TissueLyser LT, Qiagen) for 10 min at 50 Hz. Samples were centrifuged for 1 min at 13,000 g and stored at −80°C until RNA extraction.

Total cell RNA was isolated from frozen testicular tissues with the Direct-zol™ RNA MiniPrep Kits (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. Tissues were homogenized by a homogenizer at 5000 rpm for three cycles of 45 s with 15 s intervals (Minilys, Bertin Technologies, Montigny-le-Bretonneux, France) in homogenizing tubes (Tissue grinding CKMix 2 mL, Bertin Technologies). The RNA was finally eluted in 30 mL of pre-warmed (50 °C) RNase-free water. Its concentration and purity were determined by an ND-1000 Nanodrop spectrophotometer (Wilmington, DE, USA). Subsequently, the RNA was reversed transcribed into complementary DNA (cDNA) using the ImProm-II™ Reverse Transcription System (Promega, Madison, WI, USA).