Rnasezap

Isoflurane (Baxter) was used to anesthetize the mice, which were quickly decapitated afterwards. To avoid differences due to circadian gene expression all animals were prepared approximately at the same daytime (3 pm). The dissecting set and workplace were cleaned with RNaseZap® (Qiagen) to eliminate the RNases. 1.5 ml tubes were cooled in dry ice and filled with RNAlater^TM (Qiagen). The nasal dorsum was incised to gain access and isolate the nasal mucosa. To extract the olfactory bulb the skull was opened via a Y-formed cut and carefully detached. The tissue was frozen immediately at -80°C until the RNA preparation was performed.

Delta-9-tetrahydrocannabinol (∆⁹-THC) was obtained from the National Institute on Drug Abuse Drug Supply (Bethesda, MD). For all experiments, ∆⁹-THC was dissolved in 0.9% saline, 5% Cremaphor EL, and 5% ethanol (18:1:1 v/v/v) and administered intraperitoneally (IP) in an injection volume of 10 ml/kg, 60 minutes prior to testing. Doses of Δ⁹-THC were selected based on previous data obtained in our lab that resulted in a 70% maximum possible effect (%MPE) in the tail-flick assay in male mice (Henderson-Redmond et al. 2020 (link), 2021 (link)). An additional group of mice was treated with vehicle (VEH) alone to serve as a control group. VEH was prepared using 0.9% saline, 5% Cremaphor EL, and 5% ethanol (18:1:1 v/v/v) and given by IP injection of 10 ml/kg 60 minutes prior to testing. RNAse Zap, Buffer RW1, Buffer RPE, diethyl pyrocarbonate water (DEPC H₂O), Wipeout Buffer, Quantiscript^® Reverse Transcriptase (RT), Quantiscript RT Buffer, RT Primer Mix, and Rnase-free water were obtained from Qiagen, Trizol from Thermo Fisher Scientific, and chloroform from Lab Alley. The Primetime Gene Expression Master Mix, rox reference dye, and Taqman primers (CB₁, CB₂, and β-actin) are from IdT Technologies.