Department of Bioinformatics and Genomics, North Carolina Research Campus, University of North Carolina at Charlotte , Charlotte, NC , USA.
PeerJ. 2015 Apr 28;3:e919. doi: 10.7717/peerj.919. eCollection 2015.
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.
可变剪接通过改变剪接位点选择使单个基因产生多种 mRNA 异构体。在动物中，细胞类型之间的 mRNA 异构体的可变剪接非常普遍，支持细胞分化。在植物中，至少有 20%的多外显子基因发生可变剪接，但组织特异性剪接的程度和意义了解较少，部分原因是难以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，因为花粉粒仅包含两种细胞类型，并且可以在不损伤细胞的情况下大量收集。此前，我们通过比较拟南芥花粉和叶片的 RNA-Seq 数据来鉴定花粉特异性剪接模式。在这里，我们使用半定量 PCR 来验证 RNA-Seq 数据分析表明花粉和叶片之间剪接差异最大的基因中的花粉特异性剪接模式。PCR 测试证实了 9 个可变剪接模式中的 8 个，第 9 个结果不确定。在 4 个基因中，转录起始位点的变化与可变剪接同时发生。这项研究强调了低成本 PCR 检测作为验证 RNA-Seq 结果的方法的价值。
