RNA snap ™: a rapid, quantitative and inexpensive, method for isolating total RNA from bacteria

Stead, Mark B.; Agrawal, Ankit; Bowden, Katherine E.; Nasir, Rakia; Mohanty, Bijoy K.; Meagher, Richard B.; Kushner, Sidney R.

doi:10.1093/nar/gks680

Cited by 171 publications

(162 citation statements)

References 15 publications

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“…RNA was extracted from E. coli following the RNAsnap protocol for gram-negative bacteria (65). Roughly 10 8 bacterial cells were harvested by centrifugation at 16,000 × g for 30 s, the supernatant was removed by aspiration, and pelleted cells were immediately transferred to liquid nitrogen to halt transcription.…”

Section: Methodsmentioning

confidence: 99%

Conserved rates and patterns of transcription errors across bacterial growth states and lifestyles

Traverse

Ochman

2016

Proc. Natl. Acad. Sci. U.S.A.

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Errors that occur during transcription have received much less attention than the mutations that occur in DNA because transcription errors are not heritable and usually result in a very limited number of altered proteins. However, transcription error rates are typically several orders of magnitude higher than the mutation rate. Also, individual transcripts can be translated multiple times, so a single error can have substantial effects on the pool of proteins. Transcription errors can also contribute to cellular noise, thereby influencing cell survival under stressful conditions, such as starvation or antibiotic stress. Implementing a method that captures transcription errors genome-wide, we measured the rates and spectra of transcription errors in Escherichia coli and in endosymbionts for which mutation and/or substitution rates are greatly elevated over those of E. coli. Under all tested conditions, across all species, and even for different categories of RNA sequences (mRNA and rRNAs), there were no significant differences in rates of transcription errors, which ranged from 2.3 × 10−5 per nucleotide in mRNA of the endosymbiont Buchnera aphidicola to 5.2 × 10−5 per nucleotide in rRNA of the endosymbiont Carsonella ruddii. The similarity of transcription error rates in these bacterial endosymbionts to that in E. coli (4.63 × 10−5 per nucleotide) is all the more surprising given that genomic erosion has resulted in the loss of transcription fidelity factors in both Buchnera and Carsonella.

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Section: Methodsmentioning

confidence: 99%

Conserved rates and patterns of transcription errors across bacterial growth states and lifestyles

Traverse

Ochman

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…via the RNeasy kit) short RNA species tend to become lost, whereas phenol-based reagents (e.g. TRIzol) bias against longer transcripts (Stead et al, 2012). TRIzol extraction has further been reported to deliver RNA in low yield for some bacterial species (Jahn et al, 2008).…”

Section: Defining the Minimal Materials Requirements For A Dual Rna-sementioning

confidence: 99%

“…This method had been optimized to recover transcripts of all sizes, including short RNA fractions (in particular miRNAs and sRNAs) in addition to larger transcripts, and in this respect may surpass traditional column-based RNA isolation techniques (Stead et al, 2012). To this end, infected cells at a given time point were trypsinized, collected and directly lysed in 600 µL of Lysis/Binding buffer of the mirVana kit (Ambion).…”

Section: Rna Techniquesmentioning

confidence: 99%

Dual RNA-seq of pathogen and host

2012

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SummaryThe infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections.However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell's physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies.The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed "Dual RNA-seq". Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella.Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and

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“…Twentyfive and 3 mL samples were taken after 1 and 2 h for protein and RNA extraction, respectively. RNA was extracted by the RNAsnap procedure (Stead et al 2012). Protein extraction was performed by resuspending the cell pellet in 2× SDS buffer (100 mM TrisHCl pH 6.8, 200 mM DTT, 2% SDS, 0.2% bromophenol blue, and 20% glycerol) and boiling the cells for 15 min.…”

Section: Analysis Of the Expression Of Ha-tagged Ptxs And Pa5194 Varimentioning

confidence: 99%

Tet-Trap, a genetic approach to the identification of bacterial RNA thermometers: application to Pseudomonas aeruginosa

Delvillani

Sciandrone

Peano

et al. 2014

RNA

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Modulation of mRNA translatability either by trans-acting factors (proteins or sRNAs) or by in cis-acting riboregulators is widespread in bacteria and controls relevant phenotypic traits. Unfortunately, global identification of post-transcriptionally regulated genes is complicated by poor structural and functional conservation of regulatory elements and by the limitations of proteomic approaches in protein quantification. We devised a genetic system for the identification of post-transcriptionally regulated genes and we applied this system to search for Pseudomonas aeruginosa RNA thermometers, a class of regulatory RNA that modulates gene translation in response to temperature changes. As P. aeruginosa is able to thrive in a broad range of environmental conditions, genes differentially expressed at 37°C versus lower temperatures may be involved in infection and survival in the human host. We prepared a plasmid vector library with translational fusions of P. aeruginosa DNA fragments (PaDNA) inserted upstream of TIP2, a short peptide able to inactivate the Tet repressor (TetR) upon expression. The library was assayed in a streptomycin-resistant merodiploid rpsL + /rpsL31 Escherichia coli strain in which the dominant rpsL + allele, which confers streptomycin sensitivity, was repressed by TetR. PaDNA fragments conferring thermosensitive streptomycin resistance (i.e., expressing PaDNA-TIP2 fusions at 37°C, but not at 28°C) were sequenced. We identified four new putative thermosensors. Two of them were validated with conventional reporter systems in E. coli and P. aeruginosa. Interestingly, one regulates the expression of ptxS, a gene implicated in P. aeruginosa pathogenesis.

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RNA snap ™: a rapid, quantitative and inexpensive, method for isolating total RNA from bacteria

Cited by 171 publications

References 15 publications

Conserved rates and patterns of transcription errors across bacterial growth states and lifestyles

Conserved rates and patterns of transcription errors across bacterial growth states and lifestyles

Dual RNA-seq of pathogen and host

Tet-Trap, a genetic approach to the identification of bacterial RNA thermometers: application to Pseudomonas aeruginosa

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