TY - JOUR
T1 - Evaluation of mRNA Localization Using Double Barrel Scanning Ion Conductance Microscopy
AU - Nashimoto, Yuji
AU - Takahashi, Yasufumi
AU - Zhou, Yuanshu
AU - Ito, Hidenori
AU - Ida, Hiroki
AU - Ino, Kosuke
AU - Matsue, Tomokazu
AU - Shiku, Hitoshi
N1 - Publisher Copyright:
© 2016 American Chemical Society.
PY - 2016/7/26
Y1 - 2016/7/26
N2 - 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.
AB - 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.
KW - cytosol collection
KW - electrochemical syringe
KW - mRNA localization
KW - scanning ion conductance microscopy
KW - single cell analysis
KW - transcriptome
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U2 - 10.1021/acsnano.6b02753
DO - 10.1021/acsnano.6b02753
M3 - Article
AN - SCOPUS:84979881220
VL - 10
SP - 6915
EP - 6922
JO - ACS Nano
JF - ACS Nano
SN - 1936-0851
IS - 7
ER -