A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.

The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional...

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Autores principales:	Marc W Schmid, Anja Schmidt, Ulrich C Klostermeier, Matthias Barann, Philip Rosenstiel, Ueli Grossniklaus
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/08c9b9f237244bc0a84388baf343ebe3
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spelling	oai:doaj.org-article:08c9b9f237244bc0a84388baf343ebe32021-11-18T07:29:17ZA powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.1932-620310.1371/journal.pone.0029685https://doaj.org/article/08c9b9f237244bc0a84388baf343ebe32012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22291893/?tool=EBIhttps://doaj.org/toc/1932-6203The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM), linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq). As a model we used the central cell of the Arabidopsis thaliana female gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed a de novo assembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms.Marc W SchmidAnja SchmidtUlrich C KlostermeierMatthias BarannPhilip RosenstielUeli GrossniklausPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 1, p e29685 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Marc W Schmid Anja Schmidt Ulrich C Klostermeier Matthias Barann Philip Rosenstiel Ueli Grossniklaus A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.
description	The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM), linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq). As a model we used the central cell of the Arabidopsis thaliana female gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed a de novo assembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms.
format	article
author	Marc W Schmid Anja Schmidt Ulrich C Klostermeier Matthias Barann Philip Rosenstiel Ueli Grossniklaus
author_facet	Marc W Schmid Anja Schmidt Ulrich C Klostermeier Matthias Barann Philip Rosenstiel Ueli Grossniklaus
author_sort	Marc W Schmid
title	A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.
title_short	A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.
title_full	A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.
title_fullStr	A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.
title_full_unstemmed	A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.
title_sort	powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and rna sequencing.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/08c9b9f237244bc0a84388baf343ebe3
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A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.

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