Tripure isolating reagent

Diaphragm muscle samples from each experimental group (sham, CIH, CIH + APO, NOX2 KO sham, and NOX2 KO CIH; n = 6–9 per group) were examined. Samples were weighed following removal from storage at −80 °C. A general lab homogeniser (Omni-Inc., Kennesaw, GA, USA) was used to homogenise samples (20–40 mg) in Tripure Isolating reagent (Roche Diagnostics Ltd., West Sussex, UK) kept on ice. The resultant homogenates were placed on ice for 20 min with intermittent vortexing to promote cell lysis. The phenol-chloroform method was utilised to extract total RNA from homogenates, in accordance with the manufacturer’s instructions. For muscle samples, an additional chloroform wash step was performed to enhance the purity of isolated RNA. By spectrophotometry, a nanodrop 1000 (Thermo Scientific, Waltham, MA, USA) was used to assess both the quantity (ng/µL) and purity of isolated RNA (260:280 and 260:230 ratios). Using an agarose gel electrophoresis system (E-gel, Life Technologies, Carlsbad, CA, USA), the integrity of isolated RNA was assessed by visualization of distinct 18S and 28S ribosomal RNA bands.

Sternohyoid muscles from all experimental groups (sham, CIH, CIH + APO, NOX2 KO sham and NOX2 KO CIH; n = 6–9 per group) were examined. Samples were removed from storage at −80°C and immediately weighed. Samples ranging from 20 to 40 mg were homogenised on ice in Tripure Isolating Reagent (Roche Diagnostics Ltd, Burgess Hill, UK) using a general laboratory homogeniser (Omni‐Inc., Kennesaw, GA, USA). Homogenates remained on ice for 20 min with intermittent vortexing to promote cell lysis. Total RNA was isolated from homogenates using the phenol–chloroform method in accordance with the manufacturer's instructions. An additional chloroform wash step was performed during phase separation. The quantity (ng/μl) and purity of isolated RNA (260:280 and 260:230 ratios) were assessed by spectrophotometry using a Nanodrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). The integrity of isolated RNA was assessed by visualisation of distinct 18S and 28S ribosomal RNA bands using an agarose gel electrophoresis system (E‐gel, Thermo Fisher Scientific).