一种用于检测选择性剪接的分子倒置探针检测方法。
A molecular inversion probe assay for detecting alternative splicing.
机构信息
Stanford Genome Technology Center, Department of Biochemistry, Stanford University School of Medicine, Palo Alto, CA USA.
出版信息
BMC Genomics. 2010 Dec 17;11:712. doi: 10.1186/1471-2164-11-712.
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.
背景
需要一种灵敏、高通量的方法来监测基因组范围内的前体 mRNA 剪接，以了解人类细胞中可变剪接 mRNA 的范围。
结果
我们采用分子反转探针（MIPs），一种基于发夹探针的技术，用于在人类组织中对单个剪接事件进行多路复用捕获和定量。可以使用 DNA 微阵列或高通量测序来定量单个 MIP 捕获探针，这允许对每个剪接连接进行独立评估。使用我们的方法，我们成功地鉴定了 100%的阳性对照，并表明我们的可变剪接 MIP（asMIP）检测与定量 PCR 之间具有很强的相关性。
结论
asMIP 检测法提供了一种灵敏、准确和多路复用的方法来测量前体 mRNA 剪接。完全优化后，我们估计该检测法在单个反应中可容纳超过 20000 个剪接连接。这将代表现有技术的重大改进。
Zotero 插件