Direct detection of small RNAs using splinted ligation.
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.
Maroney
Patricia A
PA
Center for RNA Molecular Biology, Case Western Reserve University, School of Medicine, W127 10900 Euclid Avenue, Cleveland, Ohio 44106-4973, USA.
Chamnongpol
Sangpen
S
Souret
Frédéric
F
Nilsen
Timothy W
TW
D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D015336:Molecular Probe Techniques; D009710:Nucleotide Mapping / Q000379:methods*; D009841:Oligonucleotides / Q000378:metabolism
D035683:MicroRNAs; D009841:Oligonucleotides