title: Subtractive hybridization identifies novel differentially expressed ncRNA species in EBV-infected human B cells.
abstract: 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.
authors.affiliation: Innsbruck Biocenter, Division of Genomics and RNomics-Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria.
authors.forename: Jan
authors.initials: J
authors.lastname: Mrázek
authors.forename: Simone B
authors.initials: SB
authors.lastname: Kreutmayer
authors.forename: Friedrich A
authors.initials: FA
authors.lastname: Grässer
authors.forename: Norbert
authors.initials: N
authors.lastname: Polacek
authors.forename: Alexander
authors.initials: A
authors.lastname: Hüttenhofer
mesh_terms: D001402:B-Lymphocytes / Q000378:metabolism / Q000821:virology; D001483:Base Sequence; D015152:Blotting, Northern; D002461:Cell Line, Transformed; D002874:Chromosome Mapping; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction; D022661:RNA, Untranslated / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D022661:RNA, Untranslated; D012367:RNA, Viral