Studying bacterial gene expression directly in infected tissues is crucial for understanding host–pathogen interactions, however recovery of intact bacterial RNA in vivo remains technically challenging. Contamination with host RNA and the instability of bacterial transcripts often limit downstream applications. Here, we developed an optimized protocol for the selective isolation of Streptococcus agalactiae RNA from infected mouse peritoneal lavage fluids. The method combines differential centrifugation, selective osmotic lysis of eukaryotic cells, and mechanical disruption of bacteria using a high-frequency bead-beating system with 106 μm glass beads. This workflow maximized RNA yield and purity, while minimizing host RNA contamination. Optimization experiments established bead size, homogenization time, and bacterial input as key parameters, enabling the recovery of detectable bacterial RNA from as few as 4 × 105 CFU/mL. In vivo, RNA preparations displayed acceptable purity (A260/280 > 1.8), with moderate integrity (RINe 5.5), yet were suitable for downstream applications. As a proof of concept, quantitative RT-PCR revealed an 11-fold upregulation of the adhesin gene pbsP in vivo compared to in vitro conditions. This protocol provides a reproducible approach for obtaining bacterial RNA from host tissues, facilitating targeted gene expression studies during infection. Although not yet optimal for full transcriptomic profiling, the method provides a practical tool for investigating virulence factor expression in vivo and can be adapted to other bacterial pathogens.
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