Br cryo cooling unit

Extracted glandular tissues were trimmed of non-glandular tissue, fat, hair, and secretory exudate. Glandular tissue was excised, minced, weighed, and then homogenized in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% IGEPAL CA-630, 0.25% deoxycholate, 1 mM EDTA, pH 7.4) supplemented with cOmplete™ EDTA-Free Protease Inhibitor Cocktail (Roche Diagnostics GmbH, Switzerland). Samples were mechanically homogenized in a Bead Ruptor 24 (Omni International, USA) bead mill homogenizer in the presence of a cold airflow supplied by an OMNI BR-Cryo cooling unit to minimize heat-denaturation. Glandular tissue was subjected to 25 minutes of high-energy ceramic bead milling-assisted agitation cycles of 10 second intervals of milling followed by 15 seconds of cooling. Homogenate supernatants were collected following a brief microcentrifugation to yield clarified 10% (w/v) gland homogenates for biochemical analysis. Total protein content of clarified 10% (w/v) gland homogenates was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Homogenate protein concentrations of deer species and sex were compared by non-repeated-measures two-way ANOVA with GraphPad Prism software (GraphPad Software, USA).

Samples in RNAlater were first thawed on ice. Meanwhile, the processing chamber of a Bead Ruptor 24 (Omni International, Kennesaw, GA) was precooled to 0°C with the BR-Cryo Cooling Unit (Omni). Reinforced screw-cap microtubes (2-mL) with 2.8-mm ceramic beads (Omni) were filled with 900 μL QIAzol reagent (Qiagen). Thawed samples were transferred to QIAzol-filled bead tubes then placed on the tube carriage. Samples were homogenized for 40 seconds at 7.10 m/s and then placed on ice for 5 minutes. For complete homogenization, DRG and TG required one cycle, spinal cord and teeth required 2 cycles, and skin required 3 cycles. Afterwards, samples were transferred to sterile 1.5-mL tubes and centrifuged for 3 minutes at 8000g to remove debris. Supernatants were collected into sterile 2-mL tubes on ice before RNA isolation/purification.