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A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms
BACKGROUND: Plant development is controlled by the action of many, often connected gene regulatory networks. Differential gene expression controlled by internal and external cues is a major driver of growth and time specific differentiation in plants. Transcriptome analysis is the state-of-the-art m...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913016/ https://www.ncbi.nlm.nih.gov/pubmed/31889976 http://dx.doi.org/10.1186/s13007-019-0536-3 |
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author | Kivivirta, Kimmo Herbert, Denise Lange, Matthias Beuerlein, Knut Altmüller, Janine Becker, Annette |
author_facet | Kivivirta, Kimmo Herbert, Denise Lange, Matthias Beuerlein, Knut Altmüller, Janine Becker, Annette |
author_sort | Kivivirta, Kimmo |
collection | PubMed |
description | BACKGROUND: Plant development is controlled by the action of many, often connected gene regulatory networks. Differential gene expression controlled by internal and external cues is a major driver of growth and time specific differentiation in plants. Transcriptome analysis is the state-of-the-art method to detect spatio-temporal changes in gene expression during development. Monitoring changes in gene expression at early stages or in small plant organs and tissues requires an accurate technique of tissue isolation, which subsequently results in RNA of sufficient quality and quantity. Laser-microdissection enables such accurate dissection and collection of desired tissue from sectioned material at a microscopic level for RNA extraction and subsequent downstream analyses, such as transcriptome, proteome, genome or miRNA. RESULTS: A protocol for laser-microdissection, RNA extraction and RNA-seq was optimized and verified for three distant angiosperm species: Arabidopsis thaliana (Brassicaceae), Oryza sativa (Poaceae) and Eschscholzia californica (Papaveraceae). Previously published protocols were improved in processing speed by reducing the vacuum intensity and incubation time during tissue fixation and incubation time and cryoprotection and by applying adhesive tape. The sample preparation and sectioning of complex and heterogenous flowers produced adequate histological quality and subsequent RNA extraction from micro-dissected gynoecia reliably generated samples of sufficient quality and quantity on all species for RNA-seq. Expression analysis of growth stage specific A. thaliana and O. sativa transcriptomes showed distinct patterns of expression of chromatin remodelers on different time points of gynoecium morphogenesis from the initiation of development to post-meiotic stages. CONCLUSION: Here we describe a protocol for plant tissue preparation, cryoprotection, cryo-sectioning, laser microdissection and RNA sample preparation for Illumina sequencing of complex plant organs from three phyletically distant plant species. We are confident that this approach is widely applicable to other plant species to enable transcriptome analysis with high spatial resolution in non-model plant species. The protocol is rapid, produces high quality sections of complex organs and results in RNA of adequate quality well suited for RNA-seq approaches. We provide detailed description of each stage of sample preparation with the quality and quantity measurements as well as an analysis of generated transcriptomes. |
format | Online Article Text |
id | pubmed-6913016 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-69130162019-12-30 A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms Kivivirta, Kimmo Herbert, Denise Lange, Matthias Beuerlein, Knut Altmüller, Janine Becker, Annette Plant Methods Methodology BACKGROUND: Plant development is controlled by the action of many, often connected gene regulatory networks. Differential gene expression controlled by internal and external cues is a major driver of growth and time specific differentiation in plants. Transcriptome analysis is the state-of-the-art method to detect spatio-temporal changes in gene expression during development. Monitoring changes in gene expression at early stages or in small plant organs and tissues requires an accurate technique of tissue isolation, which subsequently results in RNA of sufficient quality and quantity. Laser-microdissection enables such accurate dissection and collection of desired tissue from sectioned material at a microscopic level for RNA extraction and subsequent downstream analyses, such as transcriptome, proteome, genome or miRNA. RESULTS: A protocol for laser-microdissection, RNA extraction and RNA-seq was optimized and verified for three distant angiosperm species: Arabidopsis thaliana (Brassicaceae), Oryza sativa (Poaceae) and Eschscholzia californica (Papaveraceae). Previously published protocols were improved in processing speed by reducing the vacuum intensity and incubation time during tissue fixation and incubation time and cryoprotection and by applying adhesive tape. The sample preparation and sectioning of complex and heterogenous flowers produced adequate histological quality and subsequent RNA extraction from micro-dissected gynoecia reliably generated samples of sufficient quality and quantity on all species for RNA-seq. Expression analysis of growth stage specific A. thaliana and O. sativa transcriptomes showed distinct patterns of expression of chromatin remodelers on different time points of gynoecium morphogenesis from the initiation of development to post-meiotic stages. CONCLUSION: Here we describe a protocol for plant tissue preparation, cryoprotection, cryo-sectioning, laser microdissection and RNA sample preparation for Illumina sequencing of complex plant organs from three phyletically distant plant species. We are confident that this approach is widely applicable to other plant species to enable transcriptome analysis with high spatial resolution in non-model plant species. The protocol is rapid, produces high quality sections of complex organs and results in RNA of adequate quality well suited for RNA-seq approaches. We provide detailed description of each stage of sample preparation with the quality and quantity measurements as well as an analysis of generated transcriptomes. BioMed Central 2019-12-16 /pmc/articles/PMC6913016/ /pubmed/31889976 http://dx.doi.org/10.1186/s13007-019-0536-3 Text en © The Author(s) 2019 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Methodology Kivivirta, Kimmo Herbert, Denise Lange, Matthias Beuerlein, Knut Altmüller, Janine Becker, Annette A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms |
title | A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms |
title_full | A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms |
title_fullStr | A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms |
title_full_unstemmed | A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms |
title_short | A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms |
title_sort | protocol for laser microdissection (lmd) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913016/ https://www.ncbi.nlm.nih.gov/pubmed/31889976 http://dx.doi.org/10.1186/s13007-019-0536-3 |
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