Dataset Information

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.

SUBMITTER: Robertson KL

PROVIDER: S-EPMC3255644 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Locked nucleic acid and flow cytometry-fluorescence in situ hybridization for the detection of bacterial small noncoding RNAs.

Robertson Kelly L KL Vora Gary J GJ

Applied and environmental microbiology 20111104 1

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 stati ...[more]

PMID: 22057868

Similar Datasets

Project description:"Candidatus Accumulibacter" is the dominant polyphosphate-accumulating organism (PAO) in denitrifying phosphorus removal (DPR) systems. In order to investigate the community structure and clade morphotypes of "Candidatus Accumulibacter" in DPR systems through flow cytometry (FCM), denitrifying phosphorus removal of almost 100% using nitrite and nitrate as the electron acceptor was achieved in sequencing batch reactors (SBRs). An optimal method of flow cytometry combined with fluorescence in situ hybridization and SYBR green I staining (FISH-staining-flow cytometry) was developed to quantify PAOs in DPR systems. By setting the width value of FCM, bacterial cells in a sludge sample were divided into three groups in different morphotypes, namely, coccus, coccobacillus, and bacillus. Average percentages that the three different PAO populations accounted for among total bacteria from SBR1 (SBR2) were 42% (45%), 14% (13%), and 4% (2%). FCM showed that the ratios of PAOs to total bacteria in the two reactors were 61% and 59%, and the quantitative PCR (qPCR) results indicated that IIC was the dominant "Candidatus Accumulibacter" clade in both denitrifying phosphorus removal systems, reaching 50% of the total "Candidatus Accumulibacter" bacteria. The subdominant clade in the reactor with nitrite as the electron acceptor was IID, accounting for 31% of the total "Candidatus Accumulibacter" bacteria. The FCM and qPCR results suggested that clades IIC and IID were both coccus, clade IIF was coccobacillus, and clade IA was bacillus. FISH analysis also indicated that PAOs were major cocci in the systems. An equivalence test of FCM-based quantification confirmed the accuracy of FISH-staining-flow cytometry, which can meet the quantitative requirements for PAOs in complex activated sludge samples.IMPORTANCE As one group of the most important functional phosphorus removal organisms, "Candidatus Accumulibacter," affiliated with the Rhodocyclus group of the Betaproteobacteria, is a widely recognized and studied PAO in the field of biological wastewater treatment. The morphotypes and population structure of clade-level "Candidatus Accumulibacter" were studied through novel FISH-staining-flow cytometry, which involved denitrifying phosphorus removal (DPR) achieving carbon and energy savings and simultaneous removal of N and P, thus inferring the different denitrifying phosphorus removal abilities of these clades. Additionally, based on this method, in situ quantification for specific polyphosphate-accumulating organisms (PAOs) enables a more efficient process and more accurate result. The establishment of FISH-staining-flow cytometry makes cell sorting of clade-level noncultivated organisms available.

Dataset Information

Locked nucleic acid and flow cytometry-fluorescence in situ hybridization for the detection of bacterial small noncoding RNAs.

Publications

Locked nucleic acid and flow cytometry-fluorescence in situ hybridization for the detection of bacterial small noncoding RNAs.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets