Detection of MicroRNAs and Assays to Monitor MicroRNA Activities In Vivo and In Vitro

MicroRNAs (miRNAs) are approx 22-nucleotide (nt) regulatory RNAs derived from endogenous genes and processed from longer (approx 70 nt, in animals) precursor RNAs (pre-miRNAs) (1 –8 ). miRNAs bind to Argonaute (Ago) proteins, such as Ago-2 (also known as eIF2C2) (6 ,9 ), and typically associate with additional proteins to form microribonucleoproteins (miRNPs) (6 ). Another class of approx 22-nt RNAs, termed short, interfering RNAs (siRNAs), is functionally related to miRNAs (10 ,11 ). siRNAs are processed from double-stranded RNA (dsRNA), bind to Argonaute proteins, and could assemble with additional proteins to form complexes termed RNA-induced silencing complexes (RISCs) (12 ). Ago-2 is the endonuclease that cleaves the RNAs targeted by miRNAs or siRNAs (13 –16 ). miRNPs and RISCs are the effector complexes that mediate translational repression or endonucleolytic cleavage of cognate mRNAs. The function of miRNAs is largely dictated by the degree of complementarity between the miRNA and its RNA target. If the complementarity is extensive, the Ago2 found in miRNPs/RISCs, cleaves a single phosphodiester bond on the target RNA, located across from the middle of the guide si/miRNA (11 ,17 ). If the complementarity is partial, the stability of the target mRNA is not affected, but its translation is repressed (18 –20 ). In both cases, near perfect complementarity of the proximal (towards the 5′ end) portion of miRNAs is required for target mRNA recognition (21 –26 ), and if the complementarity extends beyond the 10th nucleotide of the miRNA, target mRNA cleavage occurs (27 ,28 ). We describe methods for the detection of miRNAs by Northern blots, reporter-based assays that monitor miRNA-directed gene expression regulation in vivo, and an in vitro assay that recapitulates miRNA-dependent endonucleolytic cleavage of RNA targets.