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Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis

Overview of attention for article published in BMC Research Notes, December 2015
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  • Above-average Attention Score compared to outputs of the same age and source (62nd percentile)

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Title
Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis
Published in
BMC Research Notes, December 2015
DOI 10.1186/s13104-015-1726-3
Pubmed ID
Authors

Rajandas Heera, Parimannan Sivachandran, Suresh V. Chinni, Joanne Mason, Larry Croft, Manickam Ravichandran, Lee Su Yin

Abstract

Next-generation transcriptome sequencing (RNA-Seq) has become the standard practice for studying gene splicing, mutations and changes in gene expression to obtain valuable, accurate biological conclusions. However, obtaining good sequencing coverage and depth to study these is impeded by the difficulties of obtaining high quality total RNA with minimal genomic DNA contamination. With this in mind, we evaluated the performance of Phenol-free total RNA purification kit (Amresco) in comparison with TRI Reagent (MRC) and RNeasy Mini (Qiagen) for the extraction of total RNA of Pseudomonas aeruginosa which was grown in glucose-supplemented (control) and polyethylene-supplemented (growth-limiting condition) minimal medium. All three extraction methods were coupled with an in-house DNase I treatment before the yield, integrity and size distribution of the purified RNA were assessed. RNA samples extracted with the best extraction kit were then sequenced using the Illumina HiSeq 2000 platform. TRI Reagent gave the lowest yield enriched with small RNAs (sRNAs), while RNeasy gave moderate yield of good quality RNA with trace amounts of sRNAs. The Phenol-free kit, on the other hand, gave the highest yield and the best quality RNA (RIN value of 9.85 ± 0.3) with good amounts of sRNAs. Subsequent bioinformatic analysis of the sequencing data revealed that 5435 coding genes, 452 sRNAs and 7 potential novel intergenic sRNAs were detected, indicating excellent sequencing coverage across RNA size ranges. In addition, detection of low abundance transcripts and consistency of their expression profiles across replicates from the same conditions demonstrated the reproducibility of the RNA extraction technique. Amresco's Phenol-free Total RNA purification kit coupled with DNase I treatment yielded the highest quality RNAs containing good ratios of high and low molecular weight transcripts with minimal genomic DNA. These RNA extracts gave excellent non-biased sequencing coverage useful for comprehensive total transcriptome sequencing and analysis. Furthermore, our findings would be useful for those interested in studying both coding and non-coding RNAs from precious bacterial samples cultivated in growth-limiting condition, in a single sequencing run.

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The data shown below were collected from the profiles of 2 X users who shared this research output. Click here to find out more about how the information was compiled.
Mendeley readers

Mendeley readers

The data shown below were compiled from readership statistics for 123 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Malaysia 1 <1%
Spain 1 <1%
Belgium 1 <1%
Luxembourg 1 <1%
Unknown 119 97%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 27 22%
Researcher 24 20%
Student > Master 19 15%
Student > Doctoral Student 10 8%
Student > Bachelor 9 7%
Other 17 14%
Unknown 17 14%
Readers by discipline Count As %
Agricultural and Biological Sciences 37 30%
Biochemistry, Genetics and Molecular Biology 28 23%
Immunology and Microbiology 13 11%
Medicine and Dentistry 5 4%
Chemical Engineering 3 2%
Other 15 12%
Unknown 22 18%
Attention Score in Context

Attention Score in Context

This research output has an Altmetric Attention Score of 2. 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 10 December 2015.
All research outputs
#13,759,043
of 23,327,904 outputs
Outputs from BMC Research Notes
#1,727
of 4,306 outputs
Outputs of similar age
#190,502
of 391,297 outputs
Outputs of similar age from BMC Research Notes
#56
of 148 outputs
Altmetric has tracked 23,327,904 research outputs across all sources so far. This one is in the 39th percentile – i.e., 39% of other outputs scored the same or lower than it.
So far Altmetric has tracked 4,306 research outputs from this source. They typically receive a little more attention than average, with a mean Attention Score of 5.7. This one has gotten more attention than average, scoring higher than 57% of its peers.
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 391,297 tracked outputs that were published within six weeks on either side of this one in any source. This one is in the 49th percentile – i.e., 49% of its contemporaries scored the same or lower than it.
We're also able to compare this research output to 148 others from the same source and published within six weeks on either side of this one. This one has gotten more attention than average, scoring higher than 62% of its contemporaries.