You are not logged in.
Openly accessible

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

Heera, Rajandas, Sivachandran, Parimannan, Chinni, Suresh V., Mason, Joanne, Croft, Larry, Ravichandran, Manickam and Yin, Lee Su 2015, Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis., BMC research notes, vol. 8, pp. 1-11, doi: 10.1186/s13104-015-1726-3.

Attached Files
Name Description MIMEType Size Downloads
croft-efficientextraction-2015.pdf Published version application/pdf 2.34MB 1

Title Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis.
Author(s) Heera, Rajandas
Sivachandran, Parimannan
Chinni, Suresh V.
Mason, Joanne
Croft, Larry
Ravichandran, Manickam
Yin, Lee Su
Journal name BMC research notes
Volume number 8
Article ID 754
Start page 1
End page 11
Total pages 11
Publisher BioMed Central
Place of publication London, Eng.
Publication date 2015-12
ISSN 1756-0500
Keyword(s) Gene Expression Profiling
Genetic Techniques
Indicators and Reagents
Pseudomonas aeruginosa
RNA, Bacterial
Sequence Analysis, RNA
Summary BACKGROUND: 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.

RESULTS: 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.

CONCLUSIONS: 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.
Language eng
DOI 10.1186/s13104-015-1726-3
Field of Research 1199 Other Medical And Health Sciences
HERDC Research category C1.1 Refereed article in a scholarly journal
ERA Research output type C Journal article
Copyright notice ©2015, Heera et al.
Free to Read? Yes
Use Rights Creative Commons Attribution licence
Persistent URL http://hdl.handle.net/10536/DRO/DU:30106041

Document type: Journal Article
Collections: School of Life and Environmental Sciences
Open Access Collection
Connect to link resolver
 
Unless expressly stated otherwise, the copyright for items in DRO is owned by the author, with all rights reserved.

Every reasonable effort has been made to ensure that permission has been obtained for items included in DRO. If you believe that your rights have been infringed by this repository, please contact drosupport@deakin.edu.au.

Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 0 times in TR Web of Science
Scopus Citation Count Cited 0 times in Scopus
Google Scholar Search Google Scholar
Access Statistics: 8 Abstract Views, 1 File Downloads  -  Detailed Statistics
Created: Thu, 25 Jan 2018, 10:43:04 EST

Every reasonable effort has been made to ensure that permission has been obtained for items included in DRO. If you believe that your rights have been infringed by this repository, please contact drosupport@deakin.edu.au.