title: Evaluation of mRNA Localization Using Double Barrel Scanning Ion Conductance Microscopy.
abstract: Information regarding spatial mRNA localization in single cells is necessary for a better understanding of cellular functions in tissues. Here, we report a method for evaluating localization of mRNA in single cells using double-barrel scanning ion conductance microscopy (SICM). Two barrels in a nanopipette were filled with aqueous and organic electrolyte solutions and used for SICM and as an electrochemical syringe, respectively. We confirmed that the organic phase barrel could be used to collect cytosol from living cells, which is a minute but sufficient amount to assess cellular status using qPCR analysis. The water phase barrel could be used for SICM to image topography with subcellular resolution, which could be used to determine positions for analyzing mRNA expression. This system was able to evaluate mRNA localization in single cells. After puncturing the cellular membrane in a minimally invasive manner, using SICM imaging as a guide, we collected a small amount cytosol from different positions within a single cell and showed that mRNA expression depends on cellular position. In this study, we show that SICM imaging can be utilized for the analysis of mRNA localization in single cells. In addition, we fully automated the pipet movement in the XYZ-directions during the puncturing processes, making it applicable as a high-throughput system for collecting cytosol and analyzing mRNA localization.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nashimoto
authors.affiliation: Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST) , Saitama 332-0012, Japan.
authors.forename: Yasufumi
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Yuanshu
authors.initials: Y
authors.lastname: Zhou
authors.forename: Hidenori
authors.initials: H
authors.lastname: Ito
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ida
authors.forename: Kosuke
authors.initials: K
authors.lastname: Ino
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Matsue
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Shiku
keywords: cytosol collection; electrochemical syringe; mRNA localization; scanning ion conductance microscopy; single cell analysis; transcriptome