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Highly Sensitive microRNA Array Performance

ay in the same bladder, lung, and uterus total RNA samples that were used for array analysis. The mature miRNAs on the Northern blots were quantified by phosphorimaging. The hybridization assays used chemiluminescent reactions and detection. The mirVana miRNA array system accurately reflected the expression level of each miRNA as compared to the hybridization assay and Northern analysis data obtained from total RNA (Figure 4).

Figure 4. miRNA Array Quantitation. The fold change in expression of seven different miRNAs within three different pairs of tissues was determined using three independent methods of quantitation--Northern analysis, solution hybridization/digestion assay (Hyb/Dig'n), and microarray analysis. The high correlation between the three methods validates the miRNA expression data obtained from the array system.


Biological Validation

A final validation of the procedure as well as a confirmation of the biological significance of miRNAs in adult human tissues involved profiling miRNA expression in biological samples. We isolated miRNA from 26 normal human tissues. Half of each sample was used to create an miRNA pool containing miRNA from all 26 samples. The miRNAs from the pooled sample as well as each of the 26 single-tissue samples were fluorescently labeled with Cy3 or Cy5 using the mirVana miRNA Labeling Kit. Each single tissue miRNA sample was hybridized to the miRNA arrays along with a pooled sample to act as a common reference. The relative signal intensities of the single and pooled samples were compared for each miRNA. Each of the 26 normal
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