Electrophoretic analysis on cytoplasmic molecules and organelles in single cells using microfluidic fractionation

• Project/Area Number: 19H02072
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 19010:Fluid engineering-related
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: Shintaku Hirofumi 国立研究開発法人理化学研究所, 開拓研究本部, 理研白眉研究チームリーダー (80448050)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥17,420,000 (Direct Cost: ¥13,400,000、Indirect Cost: ¥4,020,000); Fiscal Year 2021: ¥5,720,000 (Direct Cost: ¥4,400,000、Indirect Cost: ¥1,320,000); Fiscal Year 2020: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2019: ¥6,630,000 (Direct Cost: ¥5,100,000、Indirect Cost: ¥1,530,000)
• Keywords: 1細胞 / RNA / 次世代シーケンス解析 / マイクロ流体 / 分子バーコード / 電気泳動 / マイクロ流体工学 / RNA-sequencing / 細胞内小器官 / 電気穿孔

Outline of Research at the Start

本研究では1細胞に含まれる顆粒や細胞内小器官を“そのまま”の状態で抽出し，その物理化学的物性解析と構成分子網羅解析を実現するマイクロ流体システムを構築する．提案するマイクロ流体システムは1細胞の捕捉および集中電場の発生を目的とした流体トラップを有する．流体トラップを挟んで電位差を与えた際に発生する集中電場を用いて細胞膜の選択的破砕と細胞質成分の高効率抽出を実現する．抽出した顆粒や細胞内小器官は過渡的等速電気泳動で濃縮後，電気泳動により物理化学特性を分析し，流路下流において分画する．分画サンプルは，次世代シーケンシングの援用により構成分子の網羅解析を行う．

Outline of Final Research Achievements

This research project aimed to develop a microfluidic system that extracts granules and cellular organelles with the native states from single cells and analyzes the physicochemical characteristics by electrophoresis and the molecular components in the subcellular structures via high-throughput sequencing by extending the SINC-seq (single-cell integrated nuclear and cytoplasmic RNA-sequencing). To this end, we developed a new microfluidic system that parallelized the 48 channels for the on-chip electrophoretic analyses and color-coded hydrogel beads for integrating the on-chip analyses and off-chip high-throughput sequencing.

Academic Significance and Societal Importance of the Research Achievements

我々の研究グループが独自に開発した細胞質-核分画技術におけるRNA流動を詳細に調べることで電気泳動がRNA長さに非依存的な抽出を実現する上で有利であることを明らかにした．このことは，電気泳動を活用した前処理技術が網羅遺伝子発現解析を実施する上で優位性を有することを意味している．また，新しいマイクロ流体システムの開発過程においてカラーコードハイドロゲルビーズ等，広範な用途への応用が期待できる要素技術の創出も達成することができた．

Research Products

• [Journal Article] Targeted permeabilization of the cell wall and extraction of charged molecules from single cells in intact plant clusters using a focused electric field (2021)
  • Author(s): Subramanian Parimalam Sangamithirai、Abdelmoez Mahmoud Nady、Tsuchida Arata、Sotta Naoyuki、Tanaka Mayuki、Kuromori Takashi、Fujiwara Toru、Hirai Masami Yokota、Yokokawa Ryuji、Oguchi Yusuke、Shintaku Hirofumi
  • Journal Title: The Analyst Volume: 146 Issue: 5 Pages: 1604-1611
  • DOI: 10.1039/d0an02163f
  • Peer Reviewed
• [Journal Article] NanoSINC-seq dissects the isoform diversity in subcellular compartments of single cells (2021)
  • Author(s): Oguchi Yusuke、Ozaki Yuka、Abdelmoez Mahmoud N.、Shintaku Hirofumi
  • Journal Title: Science Advances Volume: 7 Issue: 15
  • DOI: 10.1126/sciadv.abe0317
  • Peer Reviewed / Open Access
• [Journal Article] Different motilities of microtubules driven by kinesin-1 and kinesin-14 motors patterned on nanopillars. (2020)
  • Author(s): Taikopaul Kaneko,Ken'ya Furuta,Kazuhiro Oiwa,Hirofumi Shintaku,Hidetoshi Kotera,Ryuji Yokokawa
  • Journal Title: Science Advances Volume: 6 Issue: 4 Pages: 7413-7413
  • DOI: 10.1126/sciadv.aax7413
  • NAID: 120006779802
  • Peer Reviewed / Open Access
• [Journal Article] Distinct Kinetics in Electrophoretic Extraction of Cytoplasmic RNA from Single Cells (2019)
  • Author(s): Abdelmoez Mahmoud N.、Oguchi Yusuke、Ozaki Yuka、Yokokawa Ryuji、Kotera Hidetoshi、Shintaku Hirofumi
  • Journal Title: Analytical Chemistry Volume: 92 Issue: 1 Pages: 1485-1492
  • DOI: 10.1021/acs.analchem.9b04739
  • Peer Reviewed
• [Journal Article] Transport of microtubules according to the number and spacing of kinesin motors on gold nano-pillars (2019)
  • Author(s): Kaneko Taikopaul、Ando Suguru、Furuta Ken’ya、Oiwa Kazuhiro、Shintaku Hirofumi、Kotera Hidetoshi、Yokokawa Ryuji
  • Journal Title: Nanoscale Volume: 000 Issue: 20 Pages: 1-9
  • DOI: 10.1039/c9nr01324e
  • Peer Reviewed
• [Journal Article] Spatial Patterning of Kinesin-1 and Dynein Motor Proteins in an In Vitro Assay using Aqueous Two-Phase Systems (ATPS). (2019)
  • Author(s): Tamanna Ishrat Farhana,Tomohiro Nakagawa,Shumpei Ohara,Hirofumi Shintaku,Hidetoshi Kotera,Ryuji Yokokawa
  • Journal Title: Langmuir Volume: 35 Issue: 40 Pages: 13003-13010
  • DOI: 10.1021/acs.langmuir.9b01411
  • Peer Reviewed
• [Presentation] Multimodal profiling of single cells via on-chip electrophoresis and RNA sequencing (2021)
  • Author(s): Arata Tsuchida, Mahmoud N. Abdelmoez, Taikopaul Kaneko, Ryuji Yokokawa, and Hirofumi Shintaku
  • Organizer: The 2021 International Chemical Congress of Pacific Basin Societies (Pacifichem2021)
  • Int'l Joint Research
• [Presentation] Profiling of membrane-mechanical phenotype and gene expression in thousands of single cells (2021)
  • Author(s): Akifumi Shiomi, Taikopaul Kaneko, Kaori Nishikawa, and Hirofumi Shintaku
  • Organizer: The 2021 International Chemical Congress of Pacific Basin Societies (Pacifichem2021)
  • Int'l Joint Research
• [Presentation] Exploring transcriptional noise in subcellular compartments with on-chip electrophoretic fractionation of cytoplasmic versus nuclear RNAs (2021)
  • Author(s): Hirofumi Shintaku
  • Organizer: Human Cell Atlas Asia General Meeting 2021
  • Int'l Joint Research / Invited
• [Presentation] 核と細胞質に存在するトランスクリプトノイズの1細胞定量 (2021)
  • Author(s): 新宅 博文
  • Organizer: 情報計算化学生物学会 2021年大会
  • Invited
• [Presentation] マイクロ・ナノ電気穿孔を用いた1細胞ダイナミクス分析 (2021)
  • Author(s): 新宅 博文
  • Organizer: 日本機械学会 2021年度 年次大会
  • Invited
• [Presentation] A combined analysis of membrane-mechanical phenotyping and transcriptomics using nanoelectroporation (2021)
  • Author(s): 塩見 晃史，金子 泰洸ポール，西川 香里，新宅 博文
  • Organizer: 第59回日本生物物理学会年会
• [Presentation] １細胞電気泳動および遺伝子発現の統合解析に向けた並列マイクロ流体システムの開発 (2021)
  • Author(s): 土田 新，マハムド N. アブディルモエズ，金子 泰洸ポール，横川 隆司，新宅 博文
  • Organizer: 第12回マイクロ・ナノ工学シンポジウム
• [Presentation] トランスクリプトーム解析を組み合わせた細胞膜の機械特性計測 (2021)
  • Author(s): 塩見 晃史，金子 泰洸ポール，西川 香里，新宅 博文
  • Organizer: 日本機械学会 2021年度年次大会
• [Presentation] Electrophoretic fractionation of subcellular components of single cells for multi-omics analyses (2020)
  • Author(s): Hirofumi Shintaku
  • Organizer: Microfluidics 2020, EMBL conference
  • Int'l Joint Research / Invited
• [Presentation] ナノポアシーケンスによる1細胞細胞質およひ核トランスクリプト発現解析 (2020)
  • Author(s): 小口祐伴，尾崎由佳，Mahmoud N. Abdelmoez, 新宅博文
  • Organizer: CHEMINAS42 化学とマイクロ・ナノシステム学会 第42回研究会
• [Presentation] オンチップ電気泳動を用いた1細胞多階層相関解析 (2020)
  • Author(s): 土田 新，マハムド ナディ アブディルモエズ，横川 隆司，新宅 博文
  • Organizer: 日本機械学会 2020 年度年次大会
• [Presentation] On-chip electrokinetics dissects subcellular gene expression (2020)
  • Author(s): Hirofumi Shintaku
  • Organizer: The SPIRITS International Symposium Shaping Self-Assembled Mesoscale (Bio)Materials with Microengineering
  • Int'l Joint Research / Invited
• [Presentation] 微細構造によるマイクロスケール流れと電場制御－1細胞分析への応用 (2020)
  • Author(s): 新宅博文
  • Organizer: 第12回 技能継承フォーラム「ものづくり技能継承の現状と展望」
  • Invited
• [Presentation] Length bias-free extraction of cytoplasmic RNA from single cells by electrical lysis and electrophoresis (2019)
  • Author(s): Mahmoud N. Abdelmoez, Yusuke Oguchi, Yuka Ozaki, Ryuji Yokokawa, Hidetoshi Kotera and Hirofumi Shintaku
  • Organizer: 30th 2019 International Symposium on Micro-NanoMechatronics and Human Science
  • Int'l Joint Research
• [Presentation] Extraction of RNA from an intact single plant cell with cell wall using focused electric field (2019)
  • Author(s): Sangamithirai Subramanian Parimalam, Naoyuki Sotta, Takashi Kuromori, Toru Fujiwara, Masami Yokota Hirai, and Hirofumi Shintaku
  • Organizer: 30th 2019 International Symposium on Micro-NanoMechatronics and Human Science
  • Int'l Joint Research
• [Presentation] Integration of on-chip electrophoretic analysis on cytoplasmic RNA of single cells and high-throughput RNA-sequencing (2019)
  • Author(s): Arata Tsuchida, Ryuji Yokokawa and Hirofumi Shintaku
  • Organizer: ASME - JSME - KSME Joint Fluids Engineering Conference 2019
  • Int'l Joint Research
• [Presentation] SINC-seq: Microfluidics approach that enables correlation analyses of cytoplasmic and nuclear RNA expression of single cells (2019)
  • Author(s): Hirofumi Shintaku
  • Organizer: Gordon Research Conference, Physics and Chemistry of Microfluidics
  • Int'l Joint Research
• [Presentation] Dynamics of RNA extraction from single cells under focused electric field (2019)
  • Author(s): Mahmoud N. Abdelmoez, Yusuke Oguchi, Yuka Ozaki, Ryuji Yokokawa, Hidetoshi Kotera, Hirofumi Shintaku
  • Organizer: EMBO Workshop on Single Cell Biology
  • Int'l Joint Research
• [Presentation] Selective and direct extraction of cytoplasmic RNA from single plant cell in an intact tissue via focused electric field (2019)
  • Author(s): Sangamithirai Subramanian Parimalam, Naoyuki Sotta, Toru Fujiwara, Masami Y. Hirai, Hirofumi Shintaku
  • Organizer: EMBO Workshop on Single Cell Biology
  • Int'l Joint Research
• [Presentation] 1細胞の細胞質-核の高精度分画とRNAシーケンシング (2019)
  • Author(s): 新宅博文
  • Organizer: 第42回日本分子生物学会年会（MBSJ2019）
  • Invited
• [Presentation] 1細胞オンチップ電気泳動による細胞質-核の高精度分画 (2019)
  • Author(s): 新宅博文
  • Organizer: 第39回 キャピラリー電気泳動シンポジウム
  • Invited
• [Presentation] On-chip electrophoretic cytometry integrated with next-generation sequencing (2019)
  • Author(s): Hirofumi Shintaku
  • Organizer: International Symposium for Intelligent Ultra-precision Optics Manufacturing Technology（IUOMT 2019）
  • Int'l Joint Research / Invited
• [Presentation] オンチップ電気泳動を活用した1細胞多階層解析 (2019)
  • Author(s): 新宅博文
  • Organizer: シングルセルゲノミクス研究会
  • Invited
• [Presentation] 1細胞解析のためのオンチップ電気泳動システムの開発 (2019)
  • Author(s): 土田 新，横川 隆司，新宅 博文
  • Organizer: 第10回マイクロ・ナノ工学シンポジウム
• [Presentation] Dynamics of RNA in single cells under focused electric field (2019)
  • Author(s): Mahmoud N. Abdelmoez, Yusuke Oguchi, Yuka Ozaki, Ryuji Yokokawa, Hidetoshi Kotera, and Hirofumi Shintaku
  • Organizer: 日本機械学会 2019年度年次大会
• [Presentation] オンチップ電気泳動を用いた1細胞解析システムの開発 (2019)
  • Author(s): 土田 新，横川 隆司，新宅 博文
  • Organizer: シングルセルゲノミクス研究会2019
• [Book] シングルセルゲノミクス （第３章8. SINC-seq法による1細胞多階層解析） (2019)
  • Author(s): 渡辺亮，鈴木穣 編集 (新宅博文，小口祐伴，飯田慶）
  • Total Pages: 219
  • Publisher: 羊土社
  • ISBN: 9784758103831
• [Remarks] RIKEN Microfluidics Lab
  • URL: https://www.hshintaku.com/
• [Remarks] RIKEN Microfluidics Laboratory
  • URL: https://www.hshintaku.com/