Selective RNA extractions are required when studying bacterial gene expression within complex mixtures of pathogens and human cells, during adhesion, internalization and survival within the host. New technologies should be developed and implemented to enrich the amount of bacterial RNAs since the majority of RNAs are from the eukaryotic host cells, requiring high read depth coverage to capture the bacterial transcriptomes in dual-RNAseq studies. This will improve our understanding about bacterial adaptation to the host cell defenses, and about how they will adapt to an intracellular life. Here we present an RNA extraction protocol to selectively enrich the lowest bacterial RNA fraction from a mixture of human and bacterial cells, using Zirconium beads, with minimal RNA degradation. Zirconium beads have higher capacity to extract bacterial RNAs than glass beads after pathogen internalization. We optimized the beads size and composition for an optimal bacterial lysis and RNA extraction. The protocol was validated on two human cell lines, differentiated macrophages and osteoblasts, with either Gram-positive (Staphylococcus aureus) or -negative (Salmonella typhimurium) bacteria. Relative to other published protocols, yield of total RNA recovery was significantly improved, while host cell infection was performed with a lower bacterial inoculum. Within the host, bacterial RNA recovery yields were about six-fold lower than an RNA extraction from pure bacteria, but the quality of the RNA recovered was essentially similar. Bacterial RNA recovery was more efficient for S. aureus than for S. typhimurium, probably due to their higher protection by the Gram positive cell walls during the early step of eukaryotic cell lysis. These purified bacterial RNAs allow subsequent genes expression studies in the course of host cell-bacteria interactions.

研究病原体和人类细胞的复杂混合物中细菌基因的表达，以及在宿主体内的黏附，内在化和存活过程中，需要进行选择性RNA提取。由于大多数RNA都来自真核宿主细胞，因此需要开发和实施新技术来丰富细菌RNA的数量，在双RNAseq研究中需要高读取深度的覆盖率才能捕获细菌转录组。这将增进我们对细菌对宿主细胞防御的适应性以及它们如何适应细胞内生活的理解。在这里，我们提出了一种RNA提取方案，可以使用锆珠，从人和细菌细胞的混合物中选择性富集最低的细菌RNA馏分，而将RNA降解降至最低。病原体内化后，锆珠比玻璃珠具有更高的提取细菌RNA的能力。我们优化了珠子的大小和组成，以实现最佳的细菌裂解和RNA提取。该协议已在两种人类细胞系（分化的巨噬细胞和成骨细胞）上验证，其中任一革兰氏阳性（金黄色葡萄球菌）或阴性（鼠伤寒沙门氏菌）细菌。相对于其他已公开的方案，总RNA回收率得到了显着提高，而宿主细胞感染是在较低的细菌接种量下进行的。在宿主内，细菌RNA的回收率比从纯细菌中提取RNA的产率低约六倍，但是回收的RNA的质量基本相似。金黄色葡萄球菌的细菌RNA回收比鼠伤寒沙门氏菌更有效，这可能是由于它们在真核细胞裂解的早期受到革兰氏阳性细胞壁的更高保护。这些纯化的细菌RNA可在宿主细胞与细菌相互作用的过程中进行后续的基因表达研究。