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 50mm 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.