title: Laser-capture microdissection of developing barley seeds and cDNA array analysis of selected tissues.
abstract: Laser microdissection provides a useful method for isolating specific cell types from complex biological samples for downstream applications. In contrast to the texture of mammalian cells, most plant tissues exhibit a cell organization with hard, cellulose-containing cell walls, large vacuoles, and air spaces, thus complicating tissue preparation and extraction of macromolecules such as DNA and RNA. Especially, barley seeds show cell types with enormous differences in osmolarity (degenerating and differentiating tissues) and contain high amounts of the main storage product starch, thus requiring specific procedures for morphological preservation and RNA extraction. In this study, we report about methods allowing tissue-specific gene expression profiling of developing barley seeds. Details on aspects of tissue preparation, including fixation and embedding procedures, laser-capture microdissection, RNA isolation, and linear mRNA amplification to produce high-quality labelled probes for large-scale expression analysis are provided. Particular emphasis is placed on the fidelity of transcript data obtained by the developed methods in relation to the in vivo transcriptome.
authors.affiliation: Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany. thielj@ipk-gatersleben.de
authors.forename: Johannes
authors.initials: J
authors.lastname: Thiel
authors.forename: Diana
authors.initials: D
authors.lastname: Weier
authors.forename: Winfriede
authors.initials: W
authors.lastname: Weschke
mesh_terms: D015342:DNA Probes / Q000096:biosynthesis / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D001467:Hordeum / Q000166:cytology* / Q000235:genetics / Q000254:growth & development; D007553:Isotope Labeling / Q000379:methods; D007834:Lasers; D042282:Microdissection / Q000295:instrumentation / Q000379:methods*; D008867:Microtomy / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010761:Phosphorus Radioisotopes / Q000737:chemistry; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012639:Seeds / Q000166:cytology* / Q000235:genetics / Q000254:growth & development; D016707:Tissue Fixation / Q000379:methods
chemical_list: D015342:DNA Probes; D010761:Phosphorus Radioisotopes; D012313:RNA