title: A Bead-Based Microfluidic Approach to Integrated Single-Cell Gene Expression Analysis by Quantitative RT-PCR.
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.
authors.affiliation: Department of Mechatronics Engineering, Harbin Institute of Technology, Harbin, Heilongjiang, China ; Department of Mechanical Engineering, Columbia University, New York, NY, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Sun
authors.affiliation: Department of Mechanical Engineering, Columbia University, New York, NY, USA.
authors.forename: Tim
authors.initials: T
authors.lastname: Olsen
authors.affiliation: Department of Mechanical Engineering, Columbia University, New York, NY, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Department of Mechatronics Engineering, Harbin Institute of Technology, Harbin, Heilongjiang, China.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Tao
authors.affiliation: Center for Radiological Research, Columbia University, New York, NY, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Ponnaiya
authors.affiliation: Department of Radiation Oncology, Columbia University, New York, NY, USA.
authors.forename: Sally A
authors.initials: SA
authors.lastname: Amundson
authors.affiliation: Center for Radiological Research, Columbia University, New York, NY, USA ; Department of Radiation Oncology, Columbia University, New York, NY, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Brenner
authors.affiliation: Department of Mechanical Engineering, Columbia University, New York, NY, USA.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Lin
keywords: Microbead; Microfluidic; RT-qPCR; Single cell analysis