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Highly Sensitive microRNA Array Performance

the indicated amounts of synthetic miR-124 spiked into the sample following fractionation. The signal is determined by background subtraction, and is depicted in signal/noise units. The heat map on the graph shows the Log2(R/G Normalized Ratio) (Mean) of miR-124 from the same samples.


High Target Specificity

A number of miRNAs have very similar sequences. For example, there are eight variants of the let-7 family that differ by one to four nucleotides. Part of the development of the mirVana miRNA array system involved creating probes and hybridization and wash conditions that minimized cross-hybridization between closely related miRNAs. While we were unable to distinguish single nucleotide mismatches without significantly reducing sensitivity, we were able to identify conditions that provide specificity and sensitivity. To test specificity, our model system included seven different arrays with probes targeting 40 different miRNAs. Each of the seven arrays included a different miR-16 probe--one was completely homologous to miR-16, two had distinct single mismatches, two had double mismatches, and two had three mismatches. miRNA from human tissue was isolated, labeled, and hybridized to each array. Signal from the miR-16 element was normalized using the signal from the other forty elements on each array. The hybridization and wash conditions used with the miRNA arrays provided over 10-fold more signal for perfect matches than for miRNAs with <90% target homology (Figure 2). Identical experiments with miR-23 probes yielded similar results.

Figure 2. miRNA Array Specificity. Probe specifi
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