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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\u2009ml). 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 the investigation of host\u2013bacterial gene expression during sepsis. The DRIB protocol yielded 2.10\u20136.91\u2009\u00b5g of total RNA per clinical sample in our pilot cohort. Dual-species ribosomal RNA (rRNA) depletion and RNA-seq generated 16.6\u201324.8\u2009million filtered reads per sample, with 63\u00b17% of reads uniquely mapped to host or bacterial sequences. Host genes accounted for 51\u201368% (8.4\u201310.9\u2009million) reads, while 0.5\u20136.7% (79,496\u2013789,808 reads) mapped to bacterial genomes. Bioinformatic analysis revealed that both shared and individual transcriptional patterns were identified in host and bacterial responses, including pathways related to immune metabolism and metal-ion binding. Our optimized DRIB protocol and RNA-seq pipeline effectively captured both host and bacterial RNA transcription in clinical sepsis samples. Expanding this approach to larger cohorts and varying disease timepoints will provide crucial new insights into host\u2013bacterial gene co-expression dynamics in sepsis progression and outcomes.<\/jats:p>","DOI":"10.1099\/mgen.0.001501","type":"journal-article","created":{"date-parts":[[2025,9,18]],"date-time":"2025-09-18T16:16:23Z","timestamp":1758212183000},"source":"Crossref","is-referenced-by-count":1,"title":["Dual RNA isolation from blood: an optimized protocol for host and bacterial RNA purification for dual RNA-sequencing analysis in whole blood sepsis samples"],"prefix":"10.1099","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2670-0799","authenticated-orcid":false,"given":"Isabella Anna","family":"Joubert","sequence":"first","affiliation":[{"name":"School of Medical, Molecular and Forensic Sciences, Murdoch University, Perth, Australia"},{"name":"Personalised Medicine Centre, Murdoch University, Perth, Australia"},{"name":"Wesfarmers Centre of Vaccines and 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