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Nucleic Acids Research Advance Access published online on June 25, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn387
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Highly sensitive and specific microRNA expression profiling using BeadArray technology

Jing Chen, Jean Lozach, Eliza Wickham Garcia, Bret Barnes, Shujun Luo, Ivan Mikoulitch, Lixin Zhou, Gary Schroth and Jian-Bing Fan*

Illumina, Inc. 9885 Towne Centre Drive, San Diego, CA 92121, USA

*To whom correspondence should be addressed. Tel: 858 202 4588; Fax: 858 202 4680; Email: jfan{at}illumina.com

Received February 25, 2008. Revised June 2, 2008. Accepted June 3, 2008.

We have developed a highly sensitive, specific and reproducible method for microRNA (miRNA) expression profiling, using the BeadArrayTM 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 (R2 ≥ 0.97). The method has a 3.5–4 log (105–109 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 (R2 = 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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