A rapid method to simultaneously separate bacterial and eukaryotic RNA during infections reveals increased intracellular expression of <i>Staphylococcus aureus</i> and <i>Shigella flexneri</i> virulence factors

, which encodes a secreted peptide that binds the C1q protein in the classical complement pathway.IMPORTANCEInfectious diseases are one of the largest causes of deaths world-wide despite access to antimicrobials and vaccines. To develop new strategies to defeat microbial infections, a greater understanding of the infection process is needed such as analyzing the microbial and host responses during different stages of infection. Several microbes can invade host cells and being able to accurately monitor their and the host cells' gene expression is critical. Here, we have developed an easy and inexpensive method to reliably enrich for and separate bacterial and eukaryotic RNA after an intracellular infection. Our method is applicable to both Gram-negative and Gram-positive bacteria. Using this method, we have identified several genes to be upregulated during S. aureus infection of macrophage cells. Our data could prove useful to obtain new strategies for developing antimicrobial drugs.