Splinted Ligation Method to Detect Small RNAs

Abstract

In any cell, the number of RNA species is remarkably complex. The sizes of RNAs can vary from 20 nucleotides to several kilobases, and abundances can vary from a few to hundreds of thousands of molecules per cell. It is of obvious interest to determine the abundance and integrity of specific RNA species within these complex mixtures. This protocol describes the splinted ligation method to detect small RNAs. It relies on the ability of T4 DNA ligase to covalently join the terminal 3′-hydroxyl group of an RNA molecule to the labeled 5′-phosphate group of a DNA chain in the presence of a DNA “splint” or “bridge” oligonucleotide that is complementary to both. After ligation, the labeled small RNA, lengthened by the covalent addition of the ³²P-labeled oligonucleotide probe, is visualized by denaturing gel electrophoresis and phosphorimaging. This approach is recommended for the routine detection and quantification of specific small RNAs (e.g., microRNAs [miRNAs] and piwi-associated RNAs [piRNAs]).