35-minute protocol for high-yield RNA extraction from gram-positive & gram-negative bacteria at room temperature without toxic chemicals

Abstract RNA extraction is an essential procedure in molecular biology and biotechnology, facilitating various downstream applications such as gene expression analysis and sequencing. Traditional methods for RNA extraction often involve the use of hazardous chemicals and require labor-intensive procedures, presenting significant challenges, particularly for researchers in resource-limited settings. This study presents a modified protocol for RNA extraction from both Gram-positive and Gram-negative bacteria, adapted from an existing method used for SARS-CoV-2. The described simplified acidic lysis protocol (pH 5.0) eliminates the need for phenol, DNase, lysozyme, and cold centrifugation, thereby enhancing safety and reducing costs. Key modifications include the introduction of a boiling step to effectively lyse Gram-positive bacteria and the use of calcium chloride to precipitate RNA, ensuring high yield and purity. The protocol was evaluated on Staphylococcus aureus, Enterococcus spp., Escherichia coli, and Pseudomonas spp., demonstrating its effectiveness across various bacterial species. Results indicate that the protocol consistently produces high-quality RNA, with an average concentration of 5 µg/ml from an input of approximately OD600 0.8-1.0 of bacterial culture. This is further supported by favorable A 260/280 and A 260/230 purity ratios, and a consistent 2:1 ratio of 23 S to 16 S rRNA, indicative of intact RNA. The method is user-friendly, rapid taking only 35 min and accessible to researchers seeking a cost-effective and straightforward approach with minimal specialized equipment. This innovative approach offers a reliable and cost-effective alternative for RNA extraction, particularly benefiting researchers in developing nations.