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    Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes (2012)

    Art
    Zeitschriftenartikel / wissenschaftlicher Beitrag
    Autoren
    Sharbati, Soroush
    Sharbati, Jutta
    Hoeke, Lena
    Bohmer, Marc
    Einspanier, Ralf
    Forschungsprojekt
    SFB 852-TP B04: Untersuchungen zum Einfluss von intestinalen Faktoren wie Zink oder Mikroorganismen auf regulierende mircoRNA und Charakterisierung d. nachgeschalteten Reaktionsmechanismen
    Quelle
    BMC genomics; 13(1) — S. 23
    ISSN: 1471-2164
    Sprache
    Englisch
    Verweise
    URL (Volltext): http://edocs.fu-berlin.de/docs/receive/FUDOCS_document_000000019811
    DOI: 10.1186/1471-2164-13-23
    Pubmed: 22248082
    Kontakt
    Institut für Veterinär-Biochemie

    Oertzenweg 19 b
    14163 Berlin
    Tel.+49 30 838 62225 Fax.+49 30 838-62584
    email:biochemie@vetmed.fu-berlin.de

    Abstract / Zusammenfassung

    Small interfering and non-coding RNAs regulate gene expression across all kingdoms of life. MicroRNAs constitute an important group of metazoan small RNAs regulating development but also disease. Accordingly, in functional genomics microRNA expression analysis sheds more and more light on the dynamic regulation of gene expression in various cellular processes.

    We have developed custom RT-qPCR arrays allowing for accurate quantification of 31 small RNAs in triplicate using a 96 well format. In parallel, we provide accurate normalisation of microRNA expression data based on the quantification of 5 reference snRNAs. We have successfully employed such arrays to study microRNA regulation during human monocyte differentiation as well as Salmonella infection. Besides well-known protagonists such as miR-146 or miR-155, we identified the up-regulation of miR-21, miR-222, miR-23b, miR-24, miR-27a as well as miR-29 upon monocyte differentiation or infection, respectively.

    The provided protocol for RT-qPCR arrays enables straight-forward microRNA expression analysis. It is fully automatable, compliant with the MIQE guidelines and can be completed in only 1 day. The application of these arrays revealed microRNAs that may mediate monocyte host defence mechanisms by regulating the TGF-β signalling upon Salmonella infection. The introduced arrays are furthermore suited for customised quantification of any class of small non-coding RNAs as exemplified by snRNAs and thus provide a versatile tool for ubiquitous applications.