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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...

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Detalles Bibliográficos
Autores principales: Lin, Shengrong, Wang, Wenyi, Palm, Curtis, Davis, Ronald W, Juneau, Kara
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3022918/
https://www.ncbi.nlm.nih.gov/pubmed/21167051
http://dx.doi.org/10.1186/1471-2164-11-712
Descripción
Sumario: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.