Optimized Hot Phenol-Based RNA Extraction from Mycobacteria: A Robust Approach for Reliable Gene Expression Analysis

Mycobacterium tuberculosis (Mtb) remains a major global health threat, underscoring the need for reliable transcriptomic studies to understand its biology and drug resistance mechanisms. Such analyses depend on obtaining high-quality, high-yield RNA. Although several RNA extraction methods are available, many require expensive reagents, large culture volumes, or specialized equipment, limiting their suitability for large-scale studies, particularly in resource-constrained settings. Here, an optimized Hot Phenol based RNA extraction method specifically tailored for mycobacteria is presented. The method uses minimal culture volume and commonly available reagents to consistently yield high-quality RNA suitable for high-throughput transcriptomic applications. RNA quantity and integrity were assessed by gel electrophoresis and RNA integrity analysis (RIN), and its suitability for downstream applications was confirmed by qPCR and Qubit 4. To benchmark the performance of the optimized method, a parallel RNA extraction using TRIzol and RNeasy under identical experimental conditions was carried out, including the same Mycobacterium species, culture volume, growth phase (logarithmic and stationary), and lysis conditions. This allowed a direct comparison of yield, quality, feasibility, and cost. The optimized Hot Phenol method demonstrated comparable or improved RNA yield and quality while significantly reducing reagent cost and dependence on specialized equipment. Owing to its efficiency, reproducibility, and affordability, this protocol provides a practical alternative for large-scale gene expression and transcriptomic studies in Mtb and other mycobacterial species.