In the assay, the target miRNA molecule is hybridized with two oligos to form a RNA/DNA duplex. When the sequences are perfectly matched, they are aligned with the miRNA and the joint can be ligated with DNA ligase. A single nucleotide difference among miRNAs will block either the hybridization or the ligation. After the pair of oligos is ligated, the ligated molecules are subjected to real-time PCR analysis.