miRNAs differ from large messenger RNAs (mRNAs) in three key ways:

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Size: miRNAs are much smaller molecules with significant variability in abundance.

Coexistence: Mature miRNAs and their precursor forms (pre-miRNA) coexist, with the only difference being length.

Isoforms: Many miRNAs share similar sequences, with isoforms differing by just one or a few nucleotides.

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Due to these factors, traditional microarray techniques are not well-suited for accurate miRNA analysis. While some commercial miRNA microarrays exist, they often require tedious pre-isolation, struggle to differentiate between isoforms, or lack the sensitivity needed for detecting low-abundance miRNAs.

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In our miRNA array assay, each miRNA is targeted by two oligonucleotides that hybridize with the target miRNA, forming an RNA/DNA duplex. When the sequences perfectly match, the oligos align with the miRNA, and DNA ligase joins them together. A single nucleotide difference between miRNAs will block either the hybridization or ligation process, allowing for isoform differentiation.

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To enhance stability, stacking sequences are added, extending the oligos and their complementary counterparts. Once ligated, the DNA undergoes linear amplification via T7 transcription in the presence of biotin-UTP, producing RNA probes for array hybridization.

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Unique tag sequences are assigned to the ligation oligos to ensure that even single nucleotide differences between isoforms are easily detectable. This approach enables precise differentiation of miRNA isoforms during array hybridization.

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The procedure involves three steps:

1. Mix miRNA with provided oligos to form miRNA/oligo hybrids.

2. Select hybrids and ligate the miRNA-directed pairing of oligos into a single DNA strand.

3. Amplify the ligated DNA via T7 transcription to generate RNA probes for hybridization.