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

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 hybrid

SUBMITTER: Pall GS 

PROVIDER: S-EPMC1885651 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature