Optimised lysozyme-assisted TRIzol® extraction yields high-integrity RNA from the antibiotic-producing bacterium...

Efficient RNA extraction from Gram-positive bacteria, particularly the antibiotic-producing genus Amycolatopsis, remains challenging due to robust peptidoglycan cell walls and RNA instability. This study systematically optimised enzymatic lysis pre-treatment using lysozyme combined with TRIzol®/chloroform extraction to enhance RNA yield and integrity from Amycolatopsis lurida. We evaluated mechanical lysis (sonication, glass bead disruption) and enzymatic approaches across multiple incubation times (5, 10, 30 min). Optimal conditions (10 mg/mL lysozyme, 10-min incubation at 37 °C) achieved a 516-fold yield increase compared to TRIzol® alone (from 15.2 ± 3.3 to 7845.8 ± 447.7 ng/μL) and excellent RNA integrity (RIN 10 vs. 2.6). This protocol yielded approximately 235 μg total RNA from 15 mL exponential-phase cultures (440 ± 34.5 mg wet cell weight), sufficient for RNA sequencing and RT-qPCR applications. Mechanical lysis methods showed significant RNA degradation despite varied optimisation attempts. The optimised lysozyme-TRIzol® protocol provides a rapid (<3 h), cost-effective alternative to existing methods while achieving superior RNA quality for contemporary transcriptomic applications.