We have developed a highly sensitive, specific and reproducible method for microRNA (miRNA) expression profiling, using the 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. Currently, assay probes are designed to simultaneously analyse 735 well-annotated human miRNAs. Using this method, highly reproducible miRNA expression profiles were generated with 100-200 ng total RNA input. Furthermore, very similar expression profiles were obtained with total RNA and enriched small RNA species (R(2) >or= 0.97). 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, in conjunction with the 96-sample array matrix should prove useful for high-throughput expression profiling of miRNAs in large numbers of tissue samples.
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| http://dx.doi.org/10.1093/nar/gkn387 | DOI Listing |
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