A rapid, quantitative assay for direct detection of microRNAs and other small RNAs using splinted ligation.
This protocol describes a method that uses splinted ligation for in-solution, direct labeling of small RNAs from total RNA. The liquid phase hybridization method makes it possible to achieve sensitive, specific, and quantitative detection while eliminating a number of time-consuming and labor-intensive steps required for the standard Northern blot assay. 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 internally 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 and is significantly more simple and more sensitive than 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.
Chamnongpol
Sangpen
S
Affymetrix, Inc, Cleveland, OH, USA. sangpen_chamnongpol@affymetrix.com
Maroney
Patricia A
PA
Nilsen
Timothy W
TW
D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D012313:RNA / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification
D035683:MicroRNAs; D009841:Oligonucleotides; D012313:RNA