Ribosomal RNA Depletion for Massively Parallel Bacterial RNA-Sequencing Applications

  • Zhoutao ChenEmail author
  • Xiaoping Duan
Part of the Methods in Molecular Biology book series (MIMB, volume 733)


RNA-sequencing (RNA-Seq) is a digital display of a transcriptome using next-generation sequencing technologies and provides detailed, high-throughput view of the transcriptome. The first step in RNA-Seq is to isolate whole transcriptome from total RNA. Since large ribosomal RNA (rRNA) constitutes approximately 90% RNA species in total RNA, whole transcriptome analysis without any contamination from rRNA is very difficult using existing RNA isolation methods. RiboMinus purification method provides a novel and efficient method to isolate RNA molecules of the transcriptome devoid of large rRNA from total RNA for transcriptome analysis. It allows for whole transcriptome isolation through selective depletion of abundant rRNA molecules from total RNA. The rRNA depleted RNA fraction is termed as RiboMinus RNA fraction, which is enriched in polyadenylated RNA, nonpolyadenylated RNA, preprocessed RNA, tRNA, numerous regulatory RNA molecules, and other RNA transcripts of yet unknown function. Using RiboMinus method to isolate RiboMinus RNA results in up to 99.0% removal of 16S and 23S rRNA molecules from 0.5 to 10 μg total bacterial RNA based on Bioanalyzer analysis. It enables efficient whole transcriptome sequencing analysis without major contamination from highly abundant rRNA. Residual rRNA accounts for less than 10% of entire transcriptome based on both SOLiD and Genome Analyzer RNA-Seq data.

Key words

Transcriptome RNA-Seq Ribosomal RNA Bacteria 16S 23S Next-generation sequencing Ribominus Polyadenylation 



The authors thank Gary Bee and Byung-In Lee for their previous work on the RiboMinus method, and Dr. Jeff Chang and Dr. Nicholas Bergman for testing bacterial probe set and for providing feedback on the sequencing performance of RiboMinus RNA.


  1. 1.
    Ruan, Y., Le Ber, P., Ng, H., and Liu, E. (2004) Interrogating the transcriptome. Trends Biotechnol. 22, 23  –30.PubMedCrossRefGoogle Scholar
  2. 2.
    Cloonan, N., Forrest, A. R. R., Kolle, G., Gardiner, B. B. A., Faulkner, G. J., Brown, M. K. et al. (2008) Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nature Methods 5, 613–  619.PubMedCrossRefGoogle Scholar
  3. 3.
    Lister, R., O’Malley, R. C., Tonti-Filippini, J., Gregory, B. D., Berry, C. C., Millar, H., et al. (2008) Highly integrated dingle-base resolution maps of the epigenome in Arabidopsis. Cell 133, 523  –  536.PubMedCrossRefGoogle Scholar
  4. 4.
    Tang, F., Barbacioru, C., Wang, Y., Nordman, E., Lee, C., Xu, N., et al. (2009) mRNA-seq whole-transcriptome analysis of a single cell. Nature Methods 6, 377–  382.PubMedCrossRefGoogle Scholar
  5. 5.
    Cheung, A.L., Eberhardt, K.J., Fischetti, V.A. (1994) A method to isolate RNA from gram-positive bacteria and mycobacteria. Anal Biochem. 222, 511–514.PubMedCrossRefGoogle Scholar
  6. 6.
    Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. Z. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonucleases. Biochem. 18, 5294  –5299.CrossRefGoogle Scholar
  7. 7.
    Di Cello, F., Xie, Y., Paul-Satyaseela, M., and Kim, K. S. (2005) Approaches to bacterial RNA isolation and purification for microarray analysis of Escherichia coli K1 interaction with human brain microvascular endothelial cells. Journal of Clinical Microbiology 43,4197–  4199.PubMedCrossRefGoogle Scholar
  8. 8.
    Sarkar, N. (1997) Polyadenylation of mRNA in prokaryotes. Annu Rev Biochem 66, 173  –197.PubMedCrossRefGoogle Scholar
  9. 9.
    McTigue, P. M., Peterson, R. J., and Kahn, J. D. (2004) Sequence-dependent thermodynamic parameters for locked nucleic acid (LNA)-DNA duplex formation. Biochemistry 43, 5388  –5405.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Life TechnologiesCarlsbadUSA

Personalised recommendations