miRNA is different from large messenger RNA in three aspects; (1) miRNAs are small size molecules with quite a big difference in abundance, (2) mature miRNAs co-exist with their precursor pre-miRNA and pri-miRNA, which only differ in length, and (3) many miRNAs are very closely related in sequences, such as isoforms, only differing by one or a few nucleotides. Therefore, the conventional mircoarray technologies cannot directly be applied to analyzing these molecules. A number of miRNA microarray products are commercially available, but they are either tedious in requiring pre-isolation of microRNA, lack the discriminative power to differentiate between isoforms, or are not sensitive enough to monitor low abundant miRNAs.

In our array assay, each miRNA molecule is targeted by two oligos that hybridize with the target miRNA to form a RNA/DNA duplex. When the sequences are perfectly matched, the oligos are aligned with the miRNA and the joint can be ligated by DNA ligase (figure 1). A single nucleotide difference among miRNAs will block either the hybridization or the ligation; Thus, miRNA isoforms can be differentiated. Due to the small size of miRNA, the hybrid might not be stable; Therefore, we introduce the stacking sequences. By extending these two oligos along with their complementary oligos, the stability is increased. Once the pair of oligos is ligated, the ligated molecules are subjected to linear amplification via T7 transcription into RNA in the presence of biotin-UTP, which are used as probes for array hybridization. To differentiate each isoform, we assigned unique tag sequences to the ligation oligos, so that single nucleotide differences are converted into unique tag sequences. In this way, each isoform can be easily distinguished by array hybridization.

The assay procedure includes three steps: (1) mix the miRNA with provided oligos to form miRNA/oligo hybrids; (2) select the hybrids and remove free oligos, and ligate miRNA-directed pairing of oligos to become a single DNA; and (3) amplify the ligated DNA with T7 transcription.