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    Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot.

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    Author
    Pall, GS; Codony-Servat, C; Byrne, J; Ritchie, L; Hamilton, A
    Date
    2007
    Source Title
    Nucleic Acids Research
    Publisher
    Oxford University Press (OUP)
    University of Melbourne Author/s
    Hamilton, Andrew
    Affiliation
    Agriculture and Food Systems
    Metadata
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    Document Type
    Journal Article
    Citations
    Pall, G. S., Codony-Servat, C., Byrne, J., Ritchie, L. & Hamilton, A. (2007). Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot.. Nucleic Acids Res, 35 (8), pp.e60-. https://doi.org/10.1093/nar/gkm112.
    Access Status
    Open Access
    URI
    http://hdl.handle.net/11343/256218
    DOI
    10.1093/nar/gkm112
    Open Access at PMC
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1885651
    Abstract
    The northern blot, or RNA gel blot, is a widely used method for the discovery, validation and expression analysis of small regulatory RNA such as small interfering RNA (siRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA). Although it is straightforward and quantitative, the main disadvantage of a northern blot is that it detects such RNA less sensitively than most other approaches. We found that the standard dose of UV used in northern blots was not the most efficient at immobilizing small RNA of 20-40 nt on nylon membranes. However, increasing the dose of UV reduced the detection of miRNA by hybridization in northern blotting experiments. We discovered that using the soluble carbodiimide, EDC, to cross-link RNA to nylon membranes greatly improved the detection of small RNA by hybridization. Compared to standard UV cross-linking procedures, EDC cross-linking provided a 25-50-fold increase in the sensitivity of detection of siRNA from plants and miRNA or piRNA from mammalian cells. All types of hybridization probes tested benefited from the new cross-linking procedure. Cross-linking was dependent on a terminal phosphate and so, should be applicable to other related categories of small RNA.

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