title: Position-Specific Gene Expression Analysis Using a Microgram Dissection Method Combined with On-Bead cDNA Library Construction.
abstract: Gene expression analysis is a key technology that is used to understand living systems. Multicellular organisms, including plants, are composed of various tissues and cell types, each of which exhibits a unique gene expression pattern. However, because of their rigid cell walls, plant cells are difficult to isolate from the whole plant. Although laser dissection has been used to circumvent this problem, the plant sample needs to be fixed beforehand, which presents several problems. In the present study, we developed an alternative method to conduct highly reliable gene expression profiling. First, we assembled a dissection apparatus that used a narrow, sharpened needle to dissect out a microsample of fresh plant tissue (0.1-0.2 mm on each side) automatically from a target site within a short time frame. Then, we optimized a protocol to synthesize a high-quality cDNA library on magnetic beads using a single microsample. The cDNA library was amplified and subjected to high-throughput sequencing. In this way, a stable and reliable system was developed to conduct gene expression profiling in small regions of a plant. The system was used to analyze the gene expression patterns at successive 50 µm intervals in the shoot apex of a 4-day-old Arabidopsis seedling. Clustering analysis of the data demonstrated that two small, adjacent domains, the shoot apical meristem and the leaf primordia, were clearly distinguishable. This system should be broadly applicable in the investigation of the spatial organization of gene expression in various contexts.
authors.affiliation: Central Research Laboratory, Hitachi, Ltd., Tokyo, 185-8601, Japan hideki.kambara.se@hitachi.com tomoharu.kajiyama.jy@hitachi.com.
authors.forename: Tomoharu
authors.initials: T
authors.lastname: Kajiyama
authors.affiliation: Central Research Laboratory, Hitachi, Ltd., Tokyo, 185-8601, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Fujii
authors.affiliation: Central Research Laboratory, Hitachi, Ltd., Tokyo, 185-8601, Japan.
authors.forename: Kouji
authors.initials: K
authors.lastname: Arikawa
authors.affiliation: Central Research Laboratory, Hitachi, Ltd., Tokyo, 185-8601, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Habu
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto, 606-8502 Japan.
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Mochizuki
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto, 606-8502 Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Nagatani
authors.affiliation: Central Research Laboratory, Hitachi, Ltd., Tokyo, 185-8601, Japan hideki.kambara.se@hitachi.com tomoharu.kajiyama.jy@hitachi.com.
authors.forename: Hideki
authors.initials: H
authors.lastname: Kambara
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics; D016000:Cluster Analysis; D018548:Cotyledon / Q000235:genetics; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D018546:Hypocotyl / Q000235:genetics; D018519:Meristem / Q000235:genetics; D042282:Microdissection / Q000295:instrumentation / Q000379:methods*; D009339:Needles; D019441:Plant Epidermis / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics; D018520:Plant Shoots / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D029681:Arabidopsis Proteins
keywords: Dissection needle; Microdissection; Organ-specific transcriptome; Position-specific gene expression; Subnanogram RNA; Whole-genome transcriptome