title: A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.
abstract: 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.
authors.affiliation: Institute of Plant Biology and Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland.
authors.forename: Marc W
authors.initials: MW
authors.lastname: Schmid
authors.forename: Anja
authors.initials: A
authors.lastname: Schmidt
authors.forename: Ulrich C
authors.initials: UC
authors.lastname: Klostermeier
authors.forename: Matthias
authors.initials: M
authors.lastname: Barann
authors.forename: Philip
authors.initials: P
authors.lastname: Rosenstiel
authors.forename: Ueli
authors.initials: U
authors.lastname: Grossniklaus
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000254:growth & development; D016000:Cluster Analysis; D056890:Eukaryota / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D061346:Laser Capture Microdissection / Q000379:methods; D046228:Microarray Analysis; D056771:Ovule / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods*; D013045:Species Specificity; D014158:Transcription, Genetic; D059467:Transcriptome