Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR
- Published
- Accepted
- Subject Areas
- Molecular Biology, Plant Science
- Keywords
- RNA-Seq, Integrated Genome Browser, Arabidopsis thaliana, Pollen, Alternative Splicing, Semi-Quantitative PCR
- Copyright
- © 2014 Estrada et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ PrePrints) and either DOI or URL of the article must be cited.
- Cite this article
- 2014. Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR. PeerJ PrePrints 2:e594v1 https://doi.org/10.7287/peerj.preprints.594v1
Abstract
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 the cell wall makes it 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. For most genes, RNA-Seq and PCR-based estimates of the different splice variants were similar, highlighting the value of the low-cost PCR assay as a method of validating RNA-Seq results.[b]
Author Comment
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