title: Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides.
abstract: A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria.
authors.affiliation: Singapore MIT Alliance for Research and Technology, 1 CREATE Way, 138602, Singapore.
authors.forename: Fabian
authors.initials: F
authors.lastname: Hia
authors.affiliation: Singapore MIT Alliance for Research and Technology, 1 CREATE Way, 138602, Singapore Department of Microbiology and Immunology Programme, National University of Singapore, 117456, Singapore.
authors.forename: Yok Hian
authors.initials: YH
authors.lastname: Chionh
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Yan Ling Joy
authors.initials: YL
authors.lastname: Pang
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: DeMott
authors.affiliation: Singapore MIT Alliance for Research and Technology, 1 CREATE Way, 138602, Singapore.
authors.forename: Megan E
authors.initials: ME
authors.lastname: McBee
authors.affiliation: Singapore MIT Alliance for Research and Technology, 1 CREATE Way, 138602, Singapore Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA pcdedon@mit.edu.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Dedon
mesh_terms: D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D009163:Mycobacterium bovis / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification; D012338:RNA, Ribosomal, 23S / Q000235:genetics / Q000302:isolation & purification; D012341:RNA, Ribosomal, 5S / Q000235:genetics* / Q000302:isolation & purification; D012343:RNA, Transfer / Q000235:genetics / Q000302:isolation & purification; D022661:RNA, Untranslated / Q000235:genetics* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012263:Ribonucleosides / Q000235:genetics
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D012341:RNA, Ribosomal, 5S; D022661:RNA, Untranslated; D012263:Ribonucleosides; D012343:RNA, Transfer