A molecular inversion probe assay for detecting alternative splicing
- Equal contributors
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
BMC Genomics 2010, 11:712 doi:10.1186/1471-2164-11-712Published: 17 December 2010
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.
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.
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.