Ryan Michael C, Zeeberg Barry R, Caplen Natasha J, Cleland James A, Kahn Ari B, Liu Hongfang, Weinstein John N
Genomics & Bioinformatics Group, Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
BMC Bioinformatics. 2008 Jul 18;9:313. doi: 10.1186/1471-2105-9-313.
Over 60% of protein-coding genes in vertebrates express mRNAs that undergo alternative splicing. The resulting collection of transcript isoforms poses significant challenges for contemporary biological assays. For example, RT-PCR validation of gene expression microarray results may be unsuccessful if the two technologies target different splice variants. Effective use of sequence-based technologies requires knowledge of the specific splice variant(s) that are targeted. In addition, the critical roles of alternative splice forms in biological function and in disease suggest that assay results may be more informative if analyzed in the context of the targeted splice variant.
A number of contemporary technologies are used for analyzing transcripts or proteins. To enable investigation of the impact of splice variation on the interpretation of data derived from those technologies, we have developed SpliceCenter. SpliceCenter is a suite of user-friendly, web-based applications that includes programs for analysis of RT-PCR primer/probe sets, effectors of RNAi, microarrays, and protein-targeting technologies. Both interactive and high-throughput implementations of the tools are provided. The interactive versions of SpliceCenter tools provide visualizations of a gene's alternative transcripts and probe target positions, enabling the user to identify which splice variants are or are not targeted. The high-throughput batch versions accept user query files and provide results in tabular form. When, for example, we used SpliceCenter's batch siRNA-Check to process the Cancer Genome Anatomy Project's large-scale shRNA library, we found that only 59% of the 50,766 shRNAs in the library target all known splice variants of the target gene, 32% target some but not all, and 9% do not target any currently annotated transcript.
SpliceCenter http://discover.nci.nih.gov/splicecenter provides unique, user-friendly applications for assessing the impact of transcript variation on the design and interpretation of RT-PCR, RNAi, gene expression microarrays, antibody-based detection, and mass spectrometry proteomics. The tools are intended for use by bench biologists as well as bioinformaticists.
脊椎动物中超过60%的蛋白质编码基因表达会经历可变剪接的mRNA。由此产生的转录本异构体集合给当代生物学检测带来了重大挑战。例如，如果两种技术针对不同的剪接变体，基因表达微阵列结果的RT-PCR验证可能会失败。有效使用基于序列的技术需要了解所针对的特定剪接变体。此外，可变剪接形式在生物学功能和疾病中的关键作用表明，如果在目标剪接变体的背景下进行分析，检测结果可能会提供更多信息。
许多当代技术用于分析转录本或蛋白质。为了能够研究剪接变异对源自这些技术的数据解释的影响，我们开发了SpliceCenter。SpliceCenter是一套用户友好的基于网络的应用程序，包括用于分析RT-PCR引物/探针集、RNAi效应物、微阵列和蛋白质靶向技术的程序。提供了工具的交互式和高通量实现方式。SpliceCenter工具的交互式版本提供了基因可变转录本和探针靶标位置的可视化，使用户能够识别哪些剪接变体被或未被靶向。高通量批处理版本接受用户查询文件并以表格形式提供结果。例如，当我们使用SpliceCenter的批处理siRNA-Check来处理癌症基因组解剖计划的大规模shRNA文库时，我们发现文库中50766个shRNA中只有59%靶向目标基因的所有已知剪接变体，32%靶向一些但不是全部，9%不靶向任何当前注释的转录本。
