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Improved northern blot method for enhanced detection of small RNA

Abstract

This protocol describes an improved northern blot method that enhances detection of small RNA molecules (<40 nt) including regulatory species such as microRNA (miRNA), short-interfering RNA (siRNA) and Piwi-interacting RNA. Northern blot analysis involves the separation of RNA molecules by denaturing gel electrophoresis followed by transfer and cross-linking of the separated molecules to nylon membrane. RNA of interest is then detected by hybridization with labeled complementary nucleic acid probes. We have replaced conventional UV-cross-linking of RNA to nylon membranes with a novel, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated, chemical cross-linking step that enhances detection of small RNA by up to 50-fold. This requires no specialized equipment, is relatively inexpensive and is technically straightforward. Northern blotting can be done in 2 d, but detection of a specific RNA can vary from minutes to days. Although chemical cross-linking takes longer (15 min to 2 h) than UV cross-linking, improved sensitivity means shorter periods of exposure are required to detect signal after hybridization.

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Figure 1: Small RNA northern blotting protocol overview.
Figure 2: Enhanced detection of short RNA with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linking versus UV cross-linking.
Figure 3: Examples of RNA preparations suitable for northern blot analysis.
Figure 4: Use of γ[32P] ATP-labeled Decade markers as a control for small RNA northern blotting procedure.

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Correspondence to Andrew J Hamilton.

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Supplementary Fig. 1

Small RNA northern blotting (EDC cross-linking) (PDF 117 kb)

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Pall, G., Hamilton, A. Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3, 1077–1084 (2008). https://doi.org/10.1038/nprot.2008.67

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