title: Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.
abstract: This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis.
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou, 310058, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou, 310058, China.
authors.forename: Yun-Xia
authors.initials: YX
authors.lastname: Zhang
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou, 310058, China.
authors.forename: Wen-Wen
authors.initials: WW
authors.lastname: Liu
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou, 310058, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou, 310058, China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Fang
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou, 310058, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Yao
mesh_terms: D002460:Cell Line; D004867:Equipment Design; D006801:Humans; D035683:MicroRNAs / Q000235:genetics; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012371:Robotics; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D035683:MicroRNAs
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