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MicroRNA Profiling by Array Analysis Reveals Critical BioMarkers

ls and Methods

miRNA Array Analysis
Total RNA from tumor and normal adjacent tissue (NAT) samples was isolated using the mirVana miRNA Isolation Kit (Ambion). Each total RNA sample (20 g) was gel fractionated using an electrophoresis unit specifically designed to speed up small RNA size fractionation and isolation. miRNA fractions for each sample were recovered and fluorescently labeled with Cy3 or Cy5 (Amersham) using the mirVana miRNA Labeling Kit (Ambion). The labeled miRNAs were hybridized (14 hours) with slides arrayed with 167 distinct miRNA probes, representing all known human and mouse miRNAs. The microarrays were washed 3 x 2 min in Wash Solution (Ambion) and scanned using a GenePix 4000B (Axon). Fluorescence intensities for the Cy3- and Cy5-labeled samples for each element were normalized by total Cy3 and Cy5 signal on the arrays. The normalized signal intensity for each element was compared between the tumor and NAT samples from each pair of patient samples and expressed as a log ratio of the tumor to normal adjacent sample.

Validation of miRNA Array Results
Northern analysis--Total RNA (1 g) was fractionated using a 15% denaturing polyacrylamide gel. The RNA was transferred to a positively charged nylon membrane by electroblotting at 200 mA in 0.5X TBE for 2 hr. After drying, the Northern blot was incubated overnight in 10 ml ULTRAhyb-Oligo (Ambion) with 107 cpm of a
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