ABSTRACT
MicroRNA (miRNA) profiling is of great interest because of the significant roles these short noncoding RNA molecules play in cellular regulation. Signature profiles, usually involving several miRNAs, have also been associated with dysfunctional cellular regulation such as in cancer. Profiling miRNAs can be done using the Agilent Technologies miRNA profiling system, which is a sensitive and accurate miRNA microarray assay. The assay is based on a highly efficient labeling method linked to a novel probe design strategy. The labeling method uses a simple, single-vial approach where 100 ng of nonfractionated total RNA is directly labeled by ligation of a Cy3 labeled pCp molecule to the 3' end of the RNA. The labeled cytosine interacts with the guanidine at the 5' end of the probe which adds stability to the hybridization complex. In addition, the probes have been designed to provide both sequence and size discrimination, generally resulting in highly specific detection of closely related mature miRNAs. The labeling and probe design strategies allow for a precise and accurate measurement that spans a linear dynamic range of greater than four orders of magnitude from at least 0.2 amol to 2 fmol of miRNA and a detection limit of less than 0.1 amol. The assay works over a wide range of sample types including FFPE samples. Agilent's microarray technology is a flexible design platform allowing quick array design iterations and incorporation of the latest miRBase content.
