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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR MECHANISMS OF PATHOGENIC BACTERIA INTERACTIONS WITH PLANT SURFACES AND ENVIRONMENTAL MATRICES

Location: Environmental Microbial and Food Safety Laboratory

Title: Evaluation of ribosomal RNA removal protocols for Salmonella RNA-Seq projects

Authors
item Bhagwat, Arvind -
item Z Ying, Irene -
item Smith, Allen

Submitted to: Advances in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 17, 2013
Publication Date: October 24, 2013
Citation: Bhagwat, A., Z Ying, I., Smith, A. 2013. Evaluation of ribosomal RNA removal protocols for Salmonella RNA-Seq projects. Advances in Microbiology. 4:25-32.

Interpretive Summary: Next generation sequencing is a powerful technology. It can provide insights into physiology of food borne human pathogens, and in particular their responses to environmental stimuli, not previously attainable. The first step in this process is to isolate and enrich messenger RNA (mRNA) from total RNA of pathogenic bacteria. In Salmonella species however, mRNA constitutes less than 1% of the total RNA population while ribosomal RNA (rRNA) constitutes >98% of the total RNA. This poses a problem because there are currently no direct methods for mRNA enrichment. An alternative option is to reduce or eliminate the rRNA. We evaluated three protocols which purport to selectively eliminate rRNA. The three protocols achieved varying degrees of rRNA depletion resulting in an 8 to 1000-fold enrichment of mRNA. The optimized protocol would reduce the sequencing cost by increasing informativeness of the sequencing data. As next generation sequencing technology is applied to foodborne pathogens we need to optimize protocols in order to generate high quality data at lower cost. This information will be useful to other scientists and will accelerate the adoption of next generation sequencing analyses for RNA preparations of mouse and human pathogens.

Technical Abstract: Next generation sequencing is a powerful technology and its application to sequencing entire RNA populations of food-borne pathogens will provide valuable insights. A problem unique to prokaryotic RNA-Seq is the massive abundance of ribosomal RNA. Unlike eukaryotic messenger RNA (mRNA), bacterial mRNA species are devoid of polyadenylation at the 3’-end and thus the approach of affinity enrichment of mRNA using oligo-dT probes is not an option. Among several approaches to enrich mRNA molecules, subtractive hybridization and removal of ribosomal RNA (rRNA) has been widely used. This approach is a single-step procedure and several rRNA-depletion kits are commercially available. We evaluated three commercially available rRNA-depletion kits to determine the efficiency of rRNA removal and enrichment of mRNA from Salmonella enterica serovar Typhimurium strain SL1344. The three protocols achieved varying degrees of rRNA depletion and resulted in 8 to 1000-fold enrichment of mRNA. rRNA removal probes from two kits were unable to titrate out 23S rRNA species and showed biased and were less efficient at 3’-end of 16S rRNA. The Ribo-Zero kit was most efficient in eliminating 16S, 23S and 5S ribosomal RNA species from the transcriptome of S. enterica serovar Typhimurium strain SL1344.

Last Modified: 9/20/2014
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