An optimised bead beating RNA extraction method for tough-to-lyse gram-positive bacteria

The extraction of high-quality bacterial RNA is essential for downstream molecular biology applications, to ensure reproducible and reliable results. Extracting RNA from gram-positive bacteria is particularly challenging due to their rigid cell wall composed of multiple layers of peptidoglycan. Therefore, there is a need for an optimised method to extract high-quality RNA from lysis resistant gram-positive bacteria. The objective of this study was to compare different cell homogenisation methods and further optimise to maximise RNA yields while retaining RNA integrity. Two different cell homogenisation methods were evaluated, bead beating (glass and zirconium beads) and high-pressure lysis (syringe). The performance of the methods was evaluated based on RNA yield, purity and integrity of the purified RNA. The glass bead beating method, using three bead beating cycles outperformed all other methods tested. The optimised method significantly improved RNA yields in L. lactis (>15 fold) and in E. faecium (>6 fold) compared to non-bead beated samples while maintaining RNA integrity (RIN >7). However, the method had minimal added benefit for S. aureus with near complete cell lysis in the absence of sample homogenisation. This method can be easily integrated into existing RNA extraction methods with the addition of a bead beating step. Furthermore, the method allows for multiple sample processing, reducing time and the risk of cross-contamination associated with other methods. • Different gram-positive bacteria cell homogenisation methods were tested. • Glass bead beating method produced the most significant yields and highest integrity RNA. • The glass bead beating method was further optimised to improve RNA yields and was tested on other gram-positive bacteria. • The optimised method using three glass bead beating cycles significantly improved RNA yields for L. lactis and E. faecium .