Evaluation of Ribosomal RNA Removal Protocols for &amp;lt;i&amp;gt;Salmonella&amp;lt;/i&amp;gt; RNA-Seq Projects

Arvind A Bhagwat,Z Irene Ying,Allen Smith

doi:10.4236/aim.2014.41006

Abstract

Next generation sequencing is a powerful technology whose application in sequencing entire RNA populations (RNA-Seq) of food-borne pathogens will provide valuable insights. A problem unique to prokaryotic RNA-Seq is removal 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 enriching mRNA molecules, removal of ribosomal RNA (rRNA) by subtractive hybridization has been widely used. This approach is a single-step procedure for which several rRNA-depletion kits are commercially available. We evaluated three commercially available rRNA-depletion kits to determine their respective efficiencies of rRNA removal 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 of the three kits were unable to titrate out 23S rRNA species while removal of 16S rRNA was less efficient. 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.

Highlights

In order to gain insight into quantitative differences in gene expression, whole transcriptome sequencing (RNASeq) has become increasingly popular
Several approaches have been proposed to eliminate ribosomal RNA (rRNA) or selectively transcribe messenger RNA (mRNA) using specific probes, there is no consensus in the available literature and the published data are highly variable [7,8,9,11]
We compared three commercial kits for rRNA removal, MICROBExpress, Ribo-Zero and RiboMinus using RNA isolated from logarithmic-growth phase S. enterica serovar Typhimurium SL1344 cells

Summary

Introduction

In order to gain insight into quantitative differences in gene expression, whole transcriptome sequencing (RNASeq) has become increasingly popular. The bacterial RNA population is comprised of rRNA, tRNA, non-coding/antisense/regulatory. Oligo-dT based capture hybridization protocols are very effective in eliminating other RNA species such as rRNA, tRNA and non-coding RNA. The challenge in bacterial mRNA enrichment is that oligo-dT based protocols are of no use as polyadenylation of bacterial mRNA is very limited [3,4]. The task of bacterial mRNA enrichment becomes further complicated by the fact that >97% of bacterial total RNA is comprised of rRNA [2,5,6]. MICROB Express kit was considered as the best protocol for eliminating ribosomal RNA species [7]. Several laboratories have designed combinations of different approaches and commercial kits to eliminate rRNA species [1,8].

Objectives

Methods

Results

Conclusion