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.

中文翻译：

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在人类宿主细胞内化后从细菌病原体中选择性回收 RNA

在宿主内粘附、内化和存活期间研究病原体和人类细胞复杂混合物中的细菌基因表达时，需要选择性 RNA 提取。由于大多数 RNA 来自真核宿主细胞，因此需要开发和实施新技术以丰富细菌 RNA 的数量，需要高读取深度覆盖以捕获双 RNAseq 研究中的细菌转录组。这将提高我们对细菌对宿主细胞防御的适应以及它们将如何适应细胞内生命的理解。在这里，我们提出了一种 RNA 提取协议，使用锆珠从人类和细菌细胞的混合物中选择性地富集最低的细菌 RNA 部分，并最大限度地减少 RNA 降解。在病原体内化后，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。该协议在两种人类细胞系、分化的巨噬细胞和成骨细胞上进行了验证，革兰氏阳性（金黄色葡萄球菌）或阴性（鼠伤寒沙门氏菌）细菌。相对于其他已公布的协议，总 RNA 回收率显着提高，而宿主细胞感染是用较低的细菌接种进行的。在宿主内，细菌 RNA 的回收率比从纯细菌中提取的 RNA 低约六倍，但回收的 RNA 的质量基本相似。金黄色葡萄球菌的细菌 RNA 回收效率高于鼠伤寒沙门氏菌，可能是由于它们在真核细胞裂解的早期阶段受到革兰氏阳性细胞壁的更高保护。这些纯化的细菌 RNA 允许在宿主细胞-细菌相互作用过程中进行后续的基因表达研究。