Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR
A peer-reviewed article of this Preprint also exists.
Author and article information
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 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.
Cite this as
2015. Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR. PeerJ PrePrints 3:e594v2 https://doi.org/10.7287/peerj.preprints.594v2note This preprint is not peer-reviewed. You may wish to reference the subsequent peer-reviewed version of this article.
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This is the revised version of the manuscript submitted to PeerJ.
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Supplemental Information
Supplemental Data File 1: Spreadsheet listing well-supported, alternative splicing events with 20% or greater splicing difference between pollen and leaves
Additional Information
Competing Interests
The authors declare they have no competing interests.
Author Contributions
April D Estrada conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Nowlan H Freese analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Ivory C Blakley analyzed the data, reviewed drafts of the paper.
Ann E Loraine conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Data Deposition
The following information was supplied regarding the deposition of related data:
http://www.bitbucket.org/lorainelab/pollenas
Funding
This work was supported by the National Science Foundation Research Coordination Network for Integrative Pollen Biology (grant no. MCB-0955431) and National Science Foundation Arabidopsis 2010 (grant no. 0820371). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
