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Sun H, Olsen T, Zhu J, et al. A Bead-Based Microfluidic Approach to Integrated Single-Cell Gene Expression Analysis by Quantitative RT-PCR. RSC Adv. 2015;5(7):4886-4893doi: 10.1039/C4RA13356K.
Sun, H., Olsen, T., Zhu, J., Tao, J., Ponnaiya, B., Amundson, S. A., Brenner, D. J., & Lin, Q. (2015). A Bead-Based Microfluidic Approach to Integrated Single-Cell Gene Expression Analysis by Quantitative RT-PCR. RSC advances, 5(7), 4886-4893. https://doi.org/10.1039/C4RA13356K
Sun, Hao, et al. "A Bead-Based Microfluidic Approach to Integrated Single-Cell Gene Expression Analysis by Quantitative RT-PCR." RSC advances vol. 5,7 (2015): 4886-4893. doi: https://doi.org/10.1039/C4RA13356K
Sun H, Olsen T, Zhu J, Tao J, Ponnaiya B, Amundson SA, Brenner DJ, Lin Q. A Bead-Based Microfluidic Approach to Integrated Single-Cell Gene Expression Analysis by Quantitative RT-PCR. RSC Adv. 2015 Jan 01;5(7):4886-4893. doi: 10.1039/C4RA13356K. PMID: 25883782; PMCID: PMC4394375.
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RSC Adv. 2015 Jan 01;5(7):4886-4893. doi: 10.1039/C4RA13356K.

A Bead-Based Microfluidic Approach to Integrated Single-Cell Gene Expression Analysis by Quantitative RT-PCR.

RSC advances

Hao Sun, Tim Olsen, Jing Zhu, Jianguo Tao, Brian Ponnaiya, Sally A Amundson, David J Brenner, Qiao Lin

Affiliations

  1. Department of Mechatronics Engineering, Harbin Institute of Technology, Harbin, Heilongjiang, China ; Department of Mechanical Engineering, Columbia University, New York, NY, USA.
  2. Department of Mechanical Engineering, Columbia University, New York, NY, USA.
  3. Department of Mechatronics Engineering, Harbin Institute of Technology, Harbin, Heilongjiang, China.
  4. Center for Radiological Research, Columbia University, New York, NY, USA.
  5. Department of Radiation Oncology, Columbia University, New York, NY, USA.
  6. Center for Radiological Research, Columbia University, New York, NY, USA ; Department of Radiation Oncology, Columbia University, New York, NY, USA.

PMID: 25883782 PMCID: PMC4394375 DOI: 10.1039/C4RA13356K

Abstract

Gene expression analysis at the single-cell level is critical to understanding variations among cells in heterogeneous populations. Microfluidic reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is well suited to gene expression assays of single cells. We present a microfluidic approach that integrates all functional steps for RT-qPCR of a single cell, including isolation and lysis of the cell, as well as purification, reverse transcription and quantitative real-time PCR of messenger RNA in the cell lysate. In this approach, all reactions in the multi-step assay of a single lysed cell can be completed on microbeads, thereby simplifying the design, fabrication and operation of the microfluidic device, as well as facilitating the minimization of sample loss or contamination. In the microfluidic device, a single cell is isolated and lysed; mRNA in the cell lysate is then analyzed by RT-qPCR using primers immobilized on microbeads in a single microchamber whose temperature is controlled in closed loop via an integrated heater and temperature sensor. The utility of the approach was demonstrated by the analysis of the effects of the drug (methyl methanesulfonate, MMS) on the induction of the cyclin-dependent kinase inhibitor 1a (CDKN1A) in single human cancer cells (MCF-7), demonstrating the potential of our approach for efficient, integrated single-cell RT-qPCR for gene expression analysis.

Keywords: Microbead; Microfluidic; RT-qPCR; Single cell analysis

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