Evaluation of mRNA Localization Using Double Barrel Scanning Ion Conductance Microscopy

Yuji Nashimoto, Yasufumi Takahashi, Yuanshu Zhou, Hidenori Ito, Hiroki Ida, Kosuke Ino, Tomokazu Matsue, Hitoshi Shiku

Research output: Contribution to journalArticle

21 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)6915-6922
Number of pages8
JournalACS Nano
Volume10
Issue number7
DOIs
Publication statusPublished - 2016 Jul 26

Keywords

  • cytosol collection
  • electrochemical syringe
  • mRNA localization
  • scanning ion conductance microscopy
  • single cell analysis
  • transcriptome

ASJC Scopus subject areas

  • Materials Science(all)
  • Engineering(all)
  • Physics and Astronomy(all)

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