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Nucleic Acids Research Advance Access published online on May 3, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm244
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Subtractive hybridization identifies novel differentially expressed ncRNA species in EBV-infected human B cells

Jan Mrázek1, Simone B. Kreutmayer1, Friedrich A. Grässer2, Norbert Polacek1,* and Alexander Hüttenhofer1,*

1Innsbruck Biocenter, Division of Genomics and RNomics—Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria and and 2Institut für Mikrobiologie und Hygiene, Abteilung Virologie, Haus 47, Universitätskliniken, D-66421 Homburg/Saar, Germany

*To whom correspondence should be addressed. Tel: + 43 (0)512 9003 70250; Fax: + 43 (0)512 9003 73100; Email: alexander.huettenhofer{at}i-med.ac.at Correspondence may also be addressed to Norbert Polacek. Email: norbert.polacek{at}i-med.ac.at

Received March 7, 2007. Revised April 3, 2007. Accepted April 3, 2007.

Non-protein-coding RNAs (ncRNAs) fulfill a wide range of cellular functions from protein synthesis to regulation of gene expression. Identification of novel regulatory ncRNAs by experimental approaches commonly includes the generation of specialized cDNA libraries encoding small ncRNA species. However, such identification is severely hampered by the presence of constitutively expressed and highly abundant ‘house-keeping’ ncRNAs, such as ribosomal RNAs, small nuclear RNAs or transfer RNAs. We have developed a novel experimental strategy, designated as subtractive hybridization of ncRNA transcripts (SHORT) to specifically select and amplify novel regulatory ncRNAs, which are only expressed at certain stages or under specific growth conditions of cells. The method is based on the selective subtractive hybridization technique, formerly applied to the detection of differentially expressed mRNAs. As a model system, we applied SHORT to Epstein–Barr virus (EBV) infected human B cells. Thereby, we identified 21 novel as well as previously reported ncRNA species to be up-regulated during virus infection. Our method will serve as a powerful tool to identify novel functional ncRNAs acting as genetic switches in the regulation of fundamental cellular processes such as development, tissue differentiation or disease.


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