biostudies-literatureUnknown11(9)Joubert IANational Health and Medical Research CouncilDual RNA-sequencing (dual RNA-seq) holds significant promise for deciphering bacterial virulence mechanisms during systemic infections. However, its application in sepsis research is hindered by technical challenges, including a low bacterial burden in blood and limited sample volumes and RNA yield from vulnerable populations, such as neonates. We developed an optimized protocol [dual RNA isolation from blood (DRIB)] for simultaneous stabilization, isolation and purification of high-quality host leukocyte and bacterial RNA from low-volume whole blood samples (0.5 ml). This protocol is compatible with clinical sample collection workflows and high-throughput RNA sequencing. The feasibility of DRIB for dual RNA-seq was validated using a pilot cohort of clinical adult sepsis samples, enabling Microbial genomicshttps://www.ebi.ac.uk/biostudies/studies/S-EPMC12447629biostudies-literatureDual RNA isolation from blood: an optimized protocol for host and bacterial RNA purification for dual RNA-sequencing analysis in whole blood sepsis samples.PMC12447629APP11878092017081Litton EWang PStrunk TChopra ACurrie ARaby EJoubert IAMullally CfalseDual RNA isolation from blood: an optimized protocol for host and bacterial RNA purification for dual RNA-sequencing analysis in whole blood sepsis samples.Dual RNA-sequencing (dual RNA-seq) holds significant promise for deciphering bacterial virulence mechanisms during systemic infections. However, its application in sepsis research is hindered by technical challenges, including a low bacterial burden in blood and limited sample volumes and RNA yield from vulnerable populations, such as neonates. We developed an optimized protocol [dual RNA isolation from blood (DRIB)] for simultaneous stabilization, isolation and purification of high-quality host leukocyte and bacterial RNA from low-volume whole blood samples (0.5 ml). This protocol is compatible with clinical sample collection workflows and high-throughput RNA sequencing. The feasibility of DRIB for dual RNA-seq was validated using a pilot cohort of clinical adult sepsis samples, enabling 2025-01-01T00:00:00Z2025 Sep2026-06-03T17:29:55.269Z2026-04-30T03:07:14.842ZS-EPMC124476294096597510.1099/mgen.0.001501