Extraction of High-Quality RNA from S. aureus Internalized by Endothelial Cells.

evades antibiotic therapy and antimicrobial defenses by entering human host cells. Bacterial transcriptomic analysis represents an invaluable tool to unravel the complex interplay between host and pathogen. Therefore, the extraction of high-quality RNA from intracellular lays the foundation to acquire meaningful gene expression data. In this study, we present a novel and straightforward strategy to isolate RNA from internalized after 90 min, 24 h, and 48 h postinfection. Real-time PCR data were obtained for the target genes and , which play major roles during infection. The commonly used reference genes , , , , and were analyzed under different conditions: bacteria from culture (condition I), intracellular bacteria (condition II), and across both conditions I and II. The most stable reference genes were used for the normalization of and . Delta C (quantification cycle) values had a relatively low variability and thus demonstrated the high quality of the extracted RNA from intracellular during the early phase of infection. The established protocol allows the extraction and purification of intracellular staphylococcal RNA while minimizing the amount of host RNA in the sample. This approach can leverage reproducible gene expression data to study host-pathogen interactions.