title: Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis.
abstract: 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.
authors.affiliation: Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia. heraadaas@gmail.com.|Unit of Biochemistry, Faculty of Medicine, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia. heraadaas@gmail.com.
authors.forename: Rajandas
authors.initials: R
authors.lastname: Heera
authors.affiliation: Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia. sivachandranparimannan@gmail.com.
authors.forename: Parimannan
authors.initials: P
authors.lastname: Sivachandran
authors.affiliation: Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia. cvsureshgupta@gmail.com.
authors.forename: Suresh V
authors.initials: SV
authors.lastname: Chinni
authors.affiliation: Malaysian Genomics Resource Centre, 27-9, Level 9 Boulevard Signature Offices, 59200, Mid Valley City, Malaysia. Joanne.mason@ouh.nhs.uk.|Oxford Biomedical Research Centre, Old Road Headington Oxford, Oxfordshire, OX3 7LE, UK. Joanne.mason@ouh.nhs.uk.
authors.forename: Joanne
authors.initials: J
authors.lastname: Mason
authors.affiliation: Malaysian Genomics Resource Centre, 27-9, Level 9 Boulevard Signature Offices, 59200, Mid Valley City, Malaysia. laurence@mgrc.com.my.
authors.forename: Larry
authors.initials: L
authors.lastname: Croft
authors.affiliation: Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia. ravichandran@aimst.edu.my.
authors.forename: Manickam
authors.initials: M
authors.lastname: Ravichandran
authors.affiliation: Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia. su_yin@aimst.edu.my.
authors.forename: Lee Su
authors.initials: LS
authors.lastname: Yin
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods*; D005821:Genetic Techniques; D007202:Indicators and Reagents; D011550:Pseudomonas aeruginosa; D012329:RNA, Bacterial / Q000302:isolation & purification*; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D007202:Indicators and Reagents; D012329:RNA, Bacterial