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Open Access Methodology article

A molecular inversion probe assay for detecting alternative splicing

Shengrong Lin12, Wenyi Wang13, Curtis Palm1, Ronald W Davis1 and Kara Juneau1*

Author Affiliations

1 Stanford Genome Technology Center, Department of Biochemistry, Stanford University School of Medicine, Palo Alto, CA USA

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

3 Department of Bioinformatics and Computational Biology, Division of Quantitative Sciences, The University of Texas MD Anderson Cancer Center, 1400 Pressler St. Unit 1410, Houston, TX 77030, USA

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BMC Genomics 2010, 11:712  doi:10.1186/1471-2164-11-712

Published: 17 December 2010

Abstract

Background

A sensitive, high-throughput method for monitoring pre-mRNA splicing on a genomic scale is needed to understand the spectrum of alternatively spliced mRNA in human cells.

Results

We adapted Molecular Inversion Probes (MIPs), a padlock-probe based technology, for the multiplexed capture and quantitation of individual splice events in human tissues. Individual MIP capture probes can be quantified using either DNA microarrays or high-throughput sequencing, which permits independent assessment of each spliced junction. Using our methodology we successfully identified 100% of our positive controls and showed that there is a strong correlation between the data from our alternative splicing MIP (asMIP) assay and quantitative PCR.

Conclusion

The asMIP assay provides a sensitive, accurate and multiplexed means for measuring pre-mRNA splicing. Fully optimized, we estimate that the assay could accommodate a throughput of greater than 20,000 splice junctions in a single reaction. This would represent a significant improvement over existing technologies.