Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR.
Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR.
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作者：
Estrada AD;Freese NH;Blakley IC;Loraine AE
选择性剪接使单个基因能够通过不同的剪接位点选择产生多个mRNA亚型。在动物中，不同细胞类型之间的mRNA异构体的选择性剪接广泛存在，并支持细胞分化。在植物中，至少20%的多外显子基因是交替剪接的，但组织特异性剪接的程度和意义还不太清楚，部分原因是很难分离单一类型的细胞。花粉是研究高等植物组织特异性剪接的有用模型系统，因为花粉粒只包含两种细胞类型，并且可以在不破坏细胞的情况下大量收集。以前，我们通过比较拟南芥花粉和叶片的RNA-Seq数据来确定花粉特异的剪接模式。在这里，我们使用半定量PCR来验证花粉特异的基因之间的剪接模式，其中RNA-Seq数据分析表明，花粉和叶片之间的剪接差异最大。聚合酶链式反应检测确认了九种可选剪接模式中的八种，而第九种剪接模式的结果并不确定。在四个基因中，选择性转录起始点与选择性剪接重合。这项研究强调了低成本的聚合酶链式反应作为验证RNA-Seq结果的一种方法的价值。
Alternative splicing enables a single gene to produce multiple mRNA isoforms by varying splice site selection. In animals, alternative splicing of mRNA isoforms between cell types is widespread and supports cellular differentiation. In plants, at least 20% of multi-exon genes are alternatively spliced, but the extent and significance of tissue-specific splicing is less well understood, partly because it is difficult to isolate cells of a single type. Pollen is a useful model system to study tissue-specific splicing in higher plants because pollen grains contain only two cell types and can be collected in large amounts without damaging cells. Previously, we identified pollen-specific splicing patterns by comparing RNA-Seq data from Arabidopsis pollen and leaves. Here, we used semi-quantitative PCR to validate pollen-specific splicing patterns among genes where RNA-Seq data analysis indicated splicing was most different between pollen and leaves. PCR testing confirmed eight of nine alternative splicing patterns, and results from the ninth were inconclusive. In four genes, alternative transcriptional start sites coincided with alternative splicing. This study highlights the value of the low-cost PCR assay as a method of validating RNA-Seq results.
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作者：
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DOI：
10.1093/bioinformatics/btp472
发表时间：
2009-10-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
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