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 pre-miRNA, which only differ in length, and (3) many miRNAs are closely related and have very similar sequences, such as isoforms, which only differ by one or a few nucleotides. Therefore, conventional microarray techniques cannot be directly applied to analyze these molecules. A number of miRNA microarray products are commercially available, but they either require the tedious pre-isolation of miRNA, are unable to differentiate between isoforms, or lack the sensitivity to detect low-abundance miRNAs.

In our miRNA 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 the miRNAs will block either the hybridization or the ligation, allowing miRNA isoforms to be differentiated. Stacking sequences are added to extend the two oligos along with their complimentary oligos, which increases the stability of the hybrid. 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 ligate miRNA-directed pairing of oligos to become a single DNA; and (3) amplify the ligated DNA with T7 transcription.