Abstract
This protocol describes a method for direct labeling and detection of small RNAs present in total RNA by splinted ligation. The assay uses a small RNA-specific bridge oligonucleotide to form base pairs with the small RNA and a 5′-end-radiolabeled ligation oligonucleotide. The captured small RNA is directly labeled by ligation. Detection of the labeled small RNAs is performed by denaturing gel electrophoresis and autoradiography or phosphorimaging. This protocol has been successfully used to study expression of various classes of biological small RNAs from nanogram to microgram amounts of total RNA without an amplification step. It is significantly simpler to perform and more sensitive than either northern blotting or ribonuclease protection assays. Once the oligonucleotides have been synthesized and total RNA has been extracted, the procedure can be completed in 6 h.
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Acknowledgements
We thank Jesse Fisher for technical assistance, and Dr. John W. Chase and Christopher J. Kubu for helpful discussions.
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Case Western Reserve University is assigned intellectual property rights related to the described technology that have been and continue to be licensed in an exclusive manner to USB Corporation. Chamnongpol and Souret declare employment at USB Corporation.
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Maroney, P., Chamnongpol, S., Souret, F. et al. Direct detection of small RNAs using splinted ligation. Nat Protoc 3, 279–287 (2008). https://doi.org/10.1038/nprot.2007.530
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DOI: https://doi.org/10.1038/nprot.2007.530
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