Date: Friday 19 February, 15:30~17:30
Speaker: Dr. Mizuki Honda
Assistant Professor, Medical Innovation Center, Graduate School of Medicine, Kyoto University
Title: Development of a high depth spatial transcriptome method limited to light-irradiated areas
Location: Zoom online
Abstract:.
Gene expression analysis of multicellular individuals and organs requires the mapping of spatial location information. However, in conventional single-cell or FACS-based gene expression analysis, spatial location information cannot be reconstructed because the cells are dissected. Laser microdissection, on the other hand, records positional information but is unable to separate fragments smaller than 100 μm in diameter. We have therefore developed a novel RNA-seq method that offers both high spatial resolution and sensitivity for detecting gene expression. In this method, tissue sections are annealed with photocleavable chemically modified caged oligo DNA, and after the reverse transcription reaction, the region of interest (ROI) on the section is irradiated with light of a specific wavelength to obtain gene expression information for the ROI alone. We dropped this caged oligo onto frozen sections containing the neural tube of developing mice, and after reverse transcription, irradiated three different adjacent regions of the neural tube (illuminated area: approx. 75 μm diameter, number of cells: approx. 80). A library was then prepared and sequenced, and approximately 10,000 genes were detected in each irradiated region, indicating that the detection sensitivity of this method is very high. The expression profile information was then dimensionally compressed and clustered according to the irradiated region. In addition, 204 genes specifically expressed in each irradiated region were obtained, and their region-specific expression was actually confirmed by in situ hybridization. In other words, this method allows for sensitive and quantitative profiling of gene expression profiles restricted to the irradiated region. Moreover, since the light irradiation can be raised to the optical limit, region-specific gene expression at the submicron level is also possible. In fact, we have successfully used this technique to detect transcripts of specific organelles in cells. In this talk, we will introduce the principle and application of our novel high depth spatial transcriptome method.