title: An Optimized Method for Extracting Bacterial RNA from Mouse Skin Tissue Colonized byMycobacterium ulcerans.
abstract: Bacterial transcriptome analyses during host colonization are essential to decipher the complexity of the relationship between the bacterium and its host. RNA sequencing (RNA-seq) is a promising approach providing valuable information about bacterial adaptation, the host response and, in some cases, mutual tolerance underlying crosstalk, as recently observed in the context ofMycobacterium ulceransinfection. Buruli ulcer is caused byM. ulcerans. This neglected disease is the third most common mycobacterial disease worldwide. Without treatment,M. ulceransprovokes massive skin ulcers. A healing process may be observed in 5% of Buruli ulcer patients several months after the initiation of disease. This spontaneous healing process suggests that some hosts can counteract the development of the lesions caused byM. ulcerans. Deciphering the mechanisms involved in this process should open up new treatment possibilities. To this end, we recently developed the first mouse model for studies of the spontaneous healing process. We have shown that the healing process is based on mutual tolerance between the bacterium and its host. In this context, RNA-seq seems to be the most appropriate method for deciphering bacterial adaptation. However, due to the low bacterial load in host tissues, the isolation of mycobacterial RNA from skin tissue for RNA-seq analysis remains challenging. We developed a method for extracting and purifying mycobacterial RNA whilst minimizing the amount of host RNA in the sample. This approach was based on the extraction of bacterial RNA by a differential lysis method. The challenge in the development of this method was the choice of a lysis system favoring the removal of host RNA without damage to the bacterial cells. We made use of the thick, resistant cell wall ofM. ulceransto achieve this end.
authors.affiliation: Center for Research in Cancerology and Immunology Nantes-Angers, Institut National de la Santé et de la Recherche Médicale, Université de Nantes, Université d'AngersAngers, France; Equipe Atip-Avenir, Center for Research in Cancerology and Immunology Nantes-Angers, Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire et Université d'AngersAngers, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Robbe-Saule
authors.affiliation: Center for Research in Cancerology and Immunology Nantes-Angers, Institut National de la Santé et de la Recherche Médicale, Université de Nantes, Université d'AngersAngers, France; Equipe Atip-Avenir, Center for Research in Cancerology and Immunology Nantes-Angers, Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire et Université d'AngersAngers, France.
authors.forename: Jérémie
authors.initials: J
authors.lastname: Babonneau
authors.affiliation: Transcriptome and Epigenome Platform, Biomics, Center for Innovation and Technological Research, Institut Pasteur Paris, France.
authors.forename: Odile
authors.initials: O
authors.lastname: Sismeiro
authors.affiliation: Center for Research in Cancerology and Immunology Nantes-Angers, Institut National de la Santé et de la Recherche Médicale, Université de Nantes, Université d'AngersAngers, France; Equipe Atip-Avenir, Center for Research in Cancerology and Immunology Nantes-Angers, Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire et Université d'AngersAngers, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Marsollier
authors.affiliation: Center for Research in Cancerology and Immunology Nantes-Angers, Institut National de la Santé et de la Recherche Médicale, Université de Nantes, Université d'AngersAngers, France; Equipe Atip-Avenir, Center for Research in Cancerology and Immunology Nantes-Angers, Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire et Université d'AngersAngers, France.
authors.forename: Estelle
authors.initials: E
authors.lastname: Marion
keywords: Mycobacterium ulcerans; RNA purification; RNA-seq; cross-talk; host-bacteria interaction