Simultaneous extraction and analyses of RNA and DNA from single cells

• Project/Area Number: 26630052
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Fluid engineering
• Research Institution: Kyoto University
• Principal Investigator: SHINTAKU HIROFUMI 京都大学, 工学研究科, 助教 (80448050)
• Co-Investigator(Kenkyū-buntansha): 小寺 秀俊 京都大学, 工学研究科, 教授 (20252471)
• Research Collaborator: Santiago Juan G. Stanford University, 教授 ; NISHIKII Hidekazu Stanford University, Visiting Scholar
• Project Period (FY): 2014-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2016: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2015: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2014: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
• Keywords: マイクロ流路 / 電気泳動 / 一細胞 / RNA / DNA / シークエンシング / 電気穿孔 / 前処理 / マイクロ流体 / 動電現象 / 等速電気泳動法 / タンパク / PCR

Outline of Final Research Achievements

In this project, we have developed a novel extraction technique of RNA and DNA from single cells with electrical lysis and electrokinetics and demonstrated it with integrated analyses on RNA and DNA. The technique leverages a microfluidic system to enable selective lysis of cytoplasmic membrane in less than 200 ms and completes extraction and separation between cytoplasmic RNA and a nucleus in less than one minute. The extracted RNA and DNA molecules showed high quality to perform high-throughput sequencing analyses. We also demonstrated our techniques with integrated nuclear and cytoplasmic RNA sequencing analyses and proposed a new strategy to study RNA transport at single cell resolution.

Research Products

• [Int'l Joint Research] Stanford University(米国)
• [Int'l Joint Research] Stanford University(米国)
• [Journal Article] Protocol for microfluidic system to automate the preparation and fractionation of the nucleic acids in the cytoplasm versus nuclei of single cells (2016)
  • Author(s): Kentaro Kuriyama, Hirofumi Shintaku, and Juan G. Santiago
  • Journal Title: Bio-protocol Volume: 6(12) Issue: 12
  • DOI: 10.21769/bioprotoc.1844
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Linearisation of λDNA Molecules by Instantaneous Variation of the Trapping Electrode Voltage Inside a Micro-Channel (2015)
  • Author(s): Itsuo HANASAKI, Naoya YUKIMOTO, Satoshi UEHARA, Hirofumi SHINTAKU, and Satoyuki KAWANO
  • Journal Title: Journal of Physics D Volume: 48 Issue: 13 Pages: 135402-135402
  • DOI: 10.1088/0022-3727/48/13/135402
  • Peer Reviewed
• [Journal Article] Isotachophoresis for Fractionation and Recovery of Cytoplasmic RNA and Nucleus from Single Cells (2015)
  • Author(s): Kentaro Kuriyama, Hirofumi Shintaku, and Juan G. Santiago
  • Journal Title: Electrophoresis Volume: - Issue: 14 Pages: 1658-1662
  • DOI: 10.1002/elps.201500040
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Extraction and Fractionation of RNA and DNA from Single Cells Using Selective Lysing and Isotachophoresis (2015)
  • Author(s): Hirofumi Shintaku and Juan G. Santiago
  • Journal Title: Proc. of SPIE Volume: 9320 Pages: 93200Q-93200Q
  • DOI: 10.1117/12.2084981
• [Journal Article] Increasing Hybridization Rate and Sensitivity of Bead Based Assays Using Isotachophoresis (2014)
  • Author(s): Hirofumi Shintaku, James W. Palko, Glenn M. Sanders, and Juan G. Santiago
  • Journal Title: Angewandte Chemie International Edition Volume: 53 Issue: 50 Pages: 13813-13816
  • DOI: 10.1002/anie.201408403
  • NAID: 120005647508
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] Electrical fractionation of cytoplasmic and nuclear nucleic acids of single cells (2017)
  • Author(s): Hirofumi Shintaku
  • Organizer: Fundamentals and Applications of Microfluidic Compartmentalization
  • Place of Presentation: Okinawa, Japan
  • Year and Date: 2017-06-13
  • Int'l Joint Research / Invited
• [Presentation] Integrated nuclear and cytoplasmic RNA sequencing of single cells (2017)
  • Author(s): Mahmoud Nady Abdel-Moez Atta, Kei Iida, Yusuke Oguchi, Sotaro Uemura, Juan G. Santiago, and Hirofumi Shintaku
  • Organizer: Advances in Genome Biology and Technology (AGBT) General Meeting 2017
  • Place of Presentation: Hollywood, FL
  • Year and Date: 2017-02-13
  • Int'l Joint Research
• [Presentation] A microfluidic system for integrated analyses on nuclear and cytoplasmic nucleic acids of single cells (2017)
  • Author(s): Hirofumi Shintaku
  • Organizer: The 12th Annual IEEE International Conference on Nano/Micro Engineered and Molecular Sytems, IEEE-NEMS 2017
  • Place of Presentation: Los Angeles, CA
  • Int'l Joint Research / Invited
• [Presentation] Massively parallel quantification of miRNA in single cells via duplex-specific nuclease reaction in pico-liter wells (2016)
  • Author(s): Sangamithirai Subramanian Parimalam, Ruji Yokokawa, Hidetoshi Kotera and Hirofumi Shintaku
  • Organizer: 2016 International conference of Microfluidics, Nanofluidics, and Lab-on-a-chip
  • Place of Presentation: Dalian, China
  • Int'l Joint Research
• [Presentation] Electrical Fractionation of Cytoplasmic RNA And Nucleus From Single Cells For Parallel RNA and DNA Analyses (2016)
  • Author(s): Hirofumi Shintaku
  • Organizer: 2nd Annual Next Generation Sequencing & Single Cell Analysis USA Congress
  • Place of Presentation: Boston, MA
  • Int'l Joint Research / Invited
• [Presentation] On-Chip Electrical Lysis and Extraction of Cytoplasmic RNA and Genomic DNA from Single Cells (2015)
  • Author(s): Hirofumi Shintaku, Kentaro Kuriyama, and Juan G. Santiago
  • Organizer: 28th International Microprocesses and Nanotechnology Conference
  • Place of Presentation: Toyama, Japan
  • Year and Date: 2015-11-10
  • Int'l Joint Research / Invited
• [Presentation] 電気穿孔および等速電気泳動を用いた一細胞前処理技術におけるRNA純度計測 (2015)
  • Author(s): 畑 翔太，横川 隆司，小寺 秀俊，新宅 博文
  • Organizer: 日本機械学会2015年度年次大会
  • Place of Presentation: 札幌
  • Year and Date: 2015-09-13
• [Presentation] On-Chip Preparation System for Simultaneous Cytoplasmic RNA and Genomic DNA Analyses of Single Cells (2015)
  • Author(s): Hirofumi Shintaku and Juan G. Santiago
  • Organizer: the 3rd Annual Single Cell Analysis Investigators Meeting
  • Place of Presentation: Bethesda, MD, USA
  • Year and Date: 2015-04-20
  • Int'l Joint Research
• [Presentation] Microfluidic System for Simultaneous Analyses of RNA and DNA in Single Cells (2015)
  • Author(s): Hirofumi Shintaku
  • Organizer: Berkeley Nanosciences and Nanoengineering Institute Seminar
  • Place of Presentation: Berkeley, CA, USA
  • Year and Date: 2015-02-27
  • Invited
• [Presentation] Extraction and Fractionation of RNA and DNA from Single Cells Using Selective Lysing and Isotachophoresis (2015)
  • Author(s): Hirofumi Shintaku, and Juan G. Santiago
  • Organizer: SPIE Photonics West BiOS
  • Place of Presentation: San Francisco, CA, USA
  • Year and Date: 2015-02-07 – 2015-02-12
  • Invited
• [Presentation] Microfluidic System for Correlation Analyses of RNA and DNA in Single Cells (2014)
  • Author(s): Hirofumi Shintaku, Kentaro Kuriyama, and Juan G. Santiago
  • Organizer: Cold Spring Harbor Asia Conferences, Single Cell
  • Place of Presentation: Suzhou, China
  • Year and Date: 2014-12-08 – 2014-12-12
  • Invited
• [Presentation] Sample Preparation for Simultaneous Analysis of RNA and DNA from Single Cells Using Electrophoretic Techniques (2014)
  • Author(s): Hirofumi Shintaku
  • Organizer: 2nd Annual Single Cell Genomics & Transcriptomics Asia Congress 2014
  • Place of Presentation: Singapore
  • Year and Date: 2014-10-07 – 2014-10-08
  • Invited
• [Presentation] Development of Microfluidic System for Isolation and Analyses of RNA and DNA from Single Cells (2014)
  • Author(s): Kentaro Kuriyama, Hirofumi Shintaku, and Juan G. Santiago
  • Organizer: 2nd Annual Single Cell Genomics & Transcriptomics Asia Congress 2014
  • Place of Presentation: Singapore
  • Year and Date: 2014-10-07 – 2014-10-08
• [Presentation] Correlating DNA and RNA Amounts in Single Cells Using Selective Lysing and Isotachophoresis (2014)
  • Author(s): 6.Hirofumi Shintaku, Kentaro Kuriyama, Hidekazu Nishikii, Lewis A. Marshall, Hidetoshi Kotera, and Juan G. Santiago
  • Organizer: SCIX 2014
  • Place of Presentation: Reno, Nevada, USA
  • Year and Date: 2014-10-01
  • Invited
• [Remarks] Kyoto University Microfluidics Group
  • URL: https://sites.google.com/site/kyotouniversitymicrofluidics/home