Isolation of Fusobacterium nucleatum-Induced Host Exosomal sRNAs

In eukaryotic cells, small RNAs (sRNAs) are a class of noncoding RNAs that are less than 200 nucleotides in length and mainly classified per their biogenesis as microRNA, small interfering RNA, PIWI-interacting RNA (piRNA) or tRNA-derived small RNA (tsRNA). Increasing lines of evidence indicate that these host-derived sRNAs may contribute to the maintenance of host-microbial homeostasis through cross-kingdom gene modulation. Recent studies have shown that when challenged with Fusobacterium nucleatum, a Gram-negative, anaerobic oral pathobiont, host epithelial cells release specific exosome-borne sRNAs, particularly tsRNAs that specifically target F. nucleatum and modulate its growth. Here, we provide an experimental procedure to isolate exosomal host-derived sRNAs when challenged with F. nucleatum. In principle, this protocol can be used for any in vitro system to study bacterial host interactions.