Printing 2-Dimentional Droplet Array for Single-Cell Reverse Transcription Quantitative PCR Assay with a Microfluidic Robot

Scientific Reports - Tập 5 Số 1
Ying Zhu1, Yunxia Zhang1, Wen-Wen Liu1, Yan Ma1, Qun Fang1, Bo Yao1
1Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou, 310058, China

Tóm tắt

AbstractThis 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.

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Scientific Reports

Tập 5 Số 1

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