title: Locked nucleic acid and flow cytometry-fluorescence in situ hybridization for the detection of bacterial small noncoding RNAs.
abstract: We describe the development and testing of a high-throughput method that enables the detection of small noncoding RNAs (ncRNAs) from single bacterial cells using locked nucleic acid probes (LNA) and flow cytometry-fluorescence in situ hybridization (flow-FISH). The LNA flow-FISH method and quantitative reverse transcription-PCR (qRT-PCR) were used to monitor the expression of three ncRNAs (6S, CsrB, and TPP-2) in Vibrio campbellii ATCC BAA-1116 cultures during lag phase, mid-log phase, and stationary phase. Both LNA flow-FISH and qRT-PCR revealed that CsrB and TPP-2 were highly expressed during lag phase but markedly reduced in mid-log phase and stationary phase, whereas 6S demonstrated no to little expression during lag phase but increased thereafter. Importantly, while LNA flow-FISH and qRT-PCR demonstrated similar overall expression trends, only LNA flow-FISH, which enabled the detection of ncRNAs in individual cells as opposed to the lysate-based ensemble measurements generated by qRT-PCR, was able to capture the cell-to-cell heterogeneity in ncRNA expression. As such, this study demonstrates a new method that simultaneously enables the in situ detection of ncRNAs and the determination of gene expression heterogeneity within an isogenic bacterial population.
authors: Center for Bio/Molecular Science & Engineering, Naval Research Laboratory, Washington, DC, USA.
authors: Kelly L
authors: KL
authors: Robertson
authors: Gary J
authors: GJ
authors: Vora
mesh_terms: D002453:Cell Cycle; D005434:Flow Cytometry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009841:Oligonucleotides; D012329:RNA, Bacterial / Q000378:metabolism*; D058727:RNA, Small Untranslated / Q000032:analysis / Q000378:metabolism*; D022661:RNA, Untranslated; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014733:Vibrio / Q000235:genetics / Q000378:metabolism*
chemical_list: C411065:6S RNA; D009841:Oligonucleotides; D012329:RNA, Bacterial; D058727:RNA, Small Untranslated; D022661:RNA, Untranslated; C477371:locked nucleic acid