A molecular inversion probe assay for detecting alternative splicing
Copyright policy )A molecular inversion probe assay for detecting alternative splicing
Absract 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.
- The University of Texas MD Anderson Cancer Center United States
- Stanford University School of Medicine United States
- Stanford University United States
- The University of Texas System United States
- Illumina (United States) United States
Reverse Transcriptase Polymerase Chain Reaction, Methodology Article, High-Throughput Nucleotide Sequencing, Molecular Probe Techniques, Reproducibility of Results, Exons, Tropomyosin, QH426-470, Reference Standards, Alternative Splicing, ROC Curve, Organ Specificity, Molecular Probes, Genetics, Humans, DNA Probes, TP248.13-248.65, Biotechnology
Reverse Transcriptase Polymerase Chain Reaction, Methodology Article, High-Throughput Nucleotide Sequencing, Molecular Probe Techniques, Reproducibility of Results, Exons, Tropomyosin, QH426-470, Reference Standards, Alternative Splicing, ROC Curve, Organ Specificity, Molecular Probes, Genetics, Humans, DNA Probes, TP248.13-248.65, Biotechnology
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