Ribosomal RNA Depletion for Efficient Use of RNA‐Seq Capacity

Dominic O'Neil1, Heike Glowatz1, Martin Schlumpberger1

1 Qiagen, Hilden, Germany
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.19
DOI:  10.1002/0471142727.mb0419s103
Online Posting Date:  July, 2013
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Abstract

Ribosomal RNA (rRNA) is the most highly abundant component of RNA, comprising the majority (>80% to 90%) of the molecules present in a total RNA sample. Depletion of this rRNA fraction is desirable prior to performing an RNA‐seq reaction, so that sequencing capacity can be focused on more informative parts of the transcriptome. This unit describes an rRNA depletion method based on selective hybridization of oligonucleotides to rRNA, recognition with a hybrid‐specific antibody, and removal of the antibody‐hybrid complex on magnetic beads. Curr. Protoc. Mol. Biol. 103:4.19.1–4.19.8. © 2013 by John Wiley & Sons, Inc.

Keywords: rRNA depletion; sample preparation; RNA‐seq; next generation sequencing; transcriptome

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Depletion of rRNA from Total RNA Using Antibody‐Mediated Hybrid Capture
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Depletion of rRNA from Total RNA Using Antibody‐Mediated Hybrid Capture

  Materials
  • GeneRead rRNA depletion kit (QIAGEN, cat. no. 180211) containing:
    • Hybridization solution
    • Antibody solution (store at 2° to 8°C)
    • HMR rRNA depletion probes (store at 2° to 8°C)
    • BioMag protein G beads (store at 2° to 8°C)
    • RNase inhibitor (store at −20°C)
    • RNase‐free H 2O
    • Small spin columns
    • 2‐ml microcentrifuge tubes
  • 1 to 5 µg purified total RNA from human, mouse, or rat (purified using QIAGEN RNeasy Plus Universal kit, cat. no. 73404)
  • RNA cleanup and concentration kit (QIAGEN RNeasy MinElute cleanup kit, cat. no. 74204)
  • Heating block
  • Heater‐shaker (e.g., Eppendorf, cat. no. 5355 000.011 with thermoblock, cat. no. 5362 000.019)
  • Vortexer
  • Sterile, RNase‐free pipet tips
  • Magnetic rack or stand
  • Microcentrifuge
  • 1.5‐ml microcentrifuge tubes
NOTE: Use only RNase‐free water for all steps.
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Figures

Videos

Literature Cited

Literature Cited
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   Tariq, M.A., Kim, H.J., Jejelowo, O., and Pourmand, N. 2011. Whole‐transcriptome RNAseq analysis from minute amounts of total RNA. Nucleic Acids Res. 39:e120.
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   Wilhelm, B.T. and Landry, J‐R. 2009. RNA‐seq‐quantitative measurement of expression through massively parallel RNA‐sequencing. Methods 48:249‐257.
   Yi, H., Cho, Y.J., Won, S., Lee, J.E., Jin Yu, H., Kim, S., Schroth, G.P., Luo, S., and Chun, J. 2011. Duplex‐specific nuclease efficiently removes rRNA for prokaryotic RNA‐seq. Nucleic Acids Res. 39:e120.
   Zhulidov, P.A., Bogdanoca, E.A., Shcheglov, A.S., Vagner, L.L., Khaspekov, G.L., Kozhemyako, V.B., Matz, M.V., Meleshkevitch, E., Moroz, L.L., Lukyanov, S.A., and Shagin, D.A. 2004. Simple cDNA normalization using Kamchatka crab duplex‐specific nuclease. Nucleic Acids Res. 32:e37.
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