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 phosphor-imaging. 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.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Maroney PA,Chamnongpol S,Souret F,Nilsen TWdoi
10.1038/nprot.2007.530subject
Has Abstractpub_date
2008-01-01 00:00:00pages
279-87issue
2eissn
1754-2189issn
1750-2799pii
nprot.2007.530journal_volume
3pub_type
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