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MicroRNA Profiling by Array Analysis Reveals Critical BioMarkers

radiolabeled probe complementary to let-7c prepared with the mirVana Probe & Marker Kit (Ambion). The blot was washed 3 x 10 min at room temperature in 2X SSC, 0.5% SDS, and then 1 x 15 min at 42C in 2X SSC, 0.5% SDS. Overnight phosphorimaging using the Storm system (Amersham) revealed the indicated mature miRNAs. The process was repeated using a radiolabeled probe for 5S or 5.8S rRNA as a loading control.

Solution hybridization detection - The mirVana miRNA Detection Kit (Ambion) was used to measure the expression levels of several other miRNAs across multiple samples. Total RNA (1 g) was incubated with radiolabeled probes for several different miRNAs. Following digestion to remove probe that was not bound by target miRNA, the radiolabeled products were fractionated by denaturing polyacrylamide gel electrophoresis and quantitated using the Storm system (Amersham). Probes were prepared by in vitro transcription with the mirVana Probe Construction Kit (Ambion).


Results and Discussion

miRNA Expression Profiling by Microarray Analysis
To analyze the global expression patterns of the nearly 200 miRNA genes identified so far, we employed a novel nonisotopic detection method. To reduce the opportunity for nonspecific cross hybridization of longer mRNA, or rRNA or precursor miRNA species to individual miRNA-specific oligonucleotide probe, Ambion scient
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