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Use of RNA Immunoprecipitation Method for Determining Sinorhizobium meliloti RNA-Hfq Protein Associations In Vivo

Overview of attention for article published in Biological Procedures Online, May 2018
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Title
Use of RNA Immunoprecipitation Method for Determining Sinorhizobium meliloti RNA-Hfq Protein Associations In Vivo
Published in
Biological Procedures Online, May 2018
DOI 10.1186/s12575-018-0075-8
Pubmed ID
Authors

Mengsheng Gao, Anne Benge, Julia M. Mesa, Regina Javier, Feng-Xia Liu

Abstract

Soil bacterium Sinorhizobium meliloti (S. meliloti) forms an endosymbiotic partnership with Medicago truncatula (M. truncatula) roots which results in root nodules. The bacteria live within root nodules where they function to fix atmospheric N2 and supply the host plant with reduced nitrogen. The bacterial RNA-binding protein Hfq (Hfq) is an important regulator for the effectiveness of the nitrogen fixation. RNA immunoprecipitation (RIP) method is a powerful method for detecting the association of Hfq protein with specific RNA in cultured bacteria, yet a RIP method for bacteria living in root nodules remains to be described. A modified S. meliloti gene encoding a His-tagged Hfq protein (HfqHis) was placed under the regulation of the native Hfq gene promoter (P hfqsm). The trans produced HfqHis protein was accumulated at its nature levels during all stages of the symbiosis, allowing RNAs that associated with the given protein to be immunoprecipitated with the anti-His antibody against the protein from root nodule lysates. RNAs that associated with the protein were selectively enriched in the immunoprecipitated sample. The RNAs were recovered by a simple method using heat and subsequently analyzed by RT-PCR. The nature of PCR products was determined by DNA sequencing. Hfq association with specific RNAs can be analyzed at different conditions (e. g. young or older root nodules) and/or in wild-type versus mutant strains. This article describes the RIP method for determining Sinorhizobium meliloti RNA-Hfq associations in vivo. It is also applicable to other rhizobia living in planta, although some tissue-specific modification related to sample disruption and homogenization may be needed.

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Mendeley readers

Mendeley readers

The data shown below were compiled from readership statistics for 15 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 15 100%

Demographic breakdown

Readers by professional status Count As %
Student > Master 3 20%
Researcher 2 13%
Student > Ph. D. Student 2 13%
Unspecified 1 7%
Student > Doctoral Student 1 7%
Other 2 13%
Unknown 4 27%
Readers by discipline Count As %
Agricultural and Biological Sciences 6 40%
Biochemistry, Genetics and Molecular Biology 4 27%
Unspecified 1 7%
Unknown 4 27%
Attention Score in Context

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 11 May 2018.
All research outputs
#19,951,180
of 25,382,440 outputs
Outputs from Biological Procedures Online
#147
of 192 outputs
Outputs of similar age
#249,516
of 339,207 outputs
Outputs of similar age from Biological Procedures Online
#2
of 4 outputs
Altmetric has tracked 25,382,440 research outputs across all sources so far. This one is in the 18th percentile – i.e., 18% of other outputs scored the same or lower than it.
So far Altmetric has tracked 192 research outputs from this source. They receive a mean Attention Score of 4.6. This one is in the 19th percentile – i.e., 19% of its peers scored the same or lower than it.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 339,207 tracked outputs that were published within six weeks on either side of this one in any source. This one is in the 21st percentile – i.e., 21% of its contemporaries scored the same or lower than it.
We're also able to compare this research output to 4 others from the same source and published within six weeks on either side of this one. This one has scored higher than 2 of them.