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.
Highlights
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
All steps including cell trapping in a specially-designed microgrooves, cell lysis by heating, cDNA synthesis, PCR amplification, and real time fluorescence detection were integrated on a single chip
We present a different type of droplet system capable of achieving single-cell reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay by printing a 2-dimentional droplet array on an oil-covered microchip with a microfluidic robot[27,28]
Summary
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. To achieve direct quantification of single-cell gene expression and increase analysis throughput, White et al.[15] combined two-step reverse transcription quantitative polymerase chain reaction (RT-qPCR) with parallel liquid operation using arrays of microchambers and microvalves. We integrated all the operations required in single cell RT-qPCR assay on a droplet array system including single cell encapsulation, cell lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, which provides a novel liquidhandling mode for single cell gene expression analysis. The feasibility of the present system for single cell gene expression analysis was demonstrated in quantitative measurement of microRNAs, which plays important roles in gene regulation[31], cell proliferation, and disease development[32]
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