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Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR

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 splic...

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Autores principales: Estrada, April D., Freese, Nowlan H., Blakley, Ivory C., Loraine, Ann E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4419537/
https://www.ncbi.nlm.nih.gov/pubmed/25945312
http://dx.doi.org/10.7717/peerj.919
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author Estrada, April D.
Freese, Nowlan H.
Blakley, Ivory C.
Loraine, Ann E.
author_facet Estrada, April D.
Freese, Nowlan H.
Blakley, Ivory C.
Loraine, Ann E.
author_sort Estrada, April D.
collection PubMed
description 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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spelling pubmed-44195372015-05-05 Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR Estrada, April D. Freese, Nowlan H. Blakley, Ivory C. Loraine, Ann E. PeerJ Bioinformatics 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. PeerJ Inc. 2015-04-28 /pmc/articles/PMC4419537/ /pubmed/25945312 http://dx.doi.org/10.7717/peerj.919 Text en © 2015 Estrada et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , 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) and either DOI or URL of the article must be cited.
spellingShingle Bioinformatics
Estrada, April D.
Freese, Nowlan H.
Blakley, Ivory C.
Loraine, Ann E.
Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR
title Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR
title_full Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR
title_fullStr Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR
title_full_unstemmed Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR
title_short Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR
title_sort analysis of pollen-specific alternative splicing in arabidopsis thaliana via semi-quantitative pcr
topic Bioinformatics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4419537/
https://www.ncbi.nlm.nih.gov/pubmed/25945312
http://dx.doi.org/10.7717/peerj.919
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