Open AccessLocked Nucleic Acid and Flow Cytometry-Fluorescence In Situ Hybridization for the Detection of Bacterial Small Noncoding RNAs
Author(s) -
Kelly L. Robertson,
Gary J. Vora
Publication year - 2012
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.06399-11
Subject(s) - flow cytometry , biology , fluorescence in situ hybridization , locked nucleic acid , in situ hybridization , nucleic acid , in situ , population , gene expression , microbiology and biotechnology , real time polymerase chain reaction , rna , gene , genetics , chemistry , demography , organic chemistry , sociology , chromosome
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-fluorescencein 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) inVibrio 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 thein situ detection of ncRNAs and the determination of gene expression heterogeneity within an isogenic bacterial population.