We have developed a highly sensitive, specific, and reproducible method for microRNA (miRNA) expression profiling, using BeadArray technology. This method incorporates an enzyme-assisted specificity step, a solid-phase primer extension to distinguish between members of miRNA families. In addition, a universal PCR is used to amplify all targets prior to array hybridization. Using this method, highly reproducible miRNA expression profiles were generated with 100-200 ng total RNA input. The method has a 3.5-4 log (10(5)-10(9) molecules) dynamic range and is able to detect 1.2- to 1.3-fold differences between samples. Expression profiles generated by this method are highly comparable to those obtained with RT-PCR (R (2) = 0.85-0.90) and direct sequencing (R = 0.87-0.89). This method should prove useful for high-throughput expression profiling of miRNAs in large numbers of tissue samples.