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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.

SUBMITTER: Zhu Y

PROVIDER: S-EPMC4381353 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

Zhu Ying Y Zhang Yun-Xia YX Liu Wen-Wen WW Ma Yan Y Fang Qun Q Yao Bo B

Scientific reports 20150401

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 ...[more]

PMID: 25828383

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Project description:The use of high-throughput multiplexed screening platforms has attracted significant interest in the field of on-site disease detection and diagnostics for their capability to simultaneously interrogate single-cell responses across different populations. However, many of the current approaches are limited by the spectral overlap between tracking materials (e.g., organic dyes) and commonly used fluorophores/biochemical stains, thus restraining their applications in multiplexed studies. This work demonstrates that the downconversion emission spectra offered by rare earth (RE)-doped β-hexagonal NaYF4 nanoparticles (NPs) can be exploited to address this spectral overlap issue. Compared to organic dyes and other tracking materials where the excitation and emission is separated by tens of nanometers, RE elements have a large gap between excitation and emission which results in their spectral independence from the organic dyes. As a proof of concept, two differently doped NaYF4 NPs (europium: Eu3+, and terbium: Tb3+) were employed on a fluorescent microscopy-based droplet microfluidic trapping array to test their feasibility as spectrally independent droplet trackers. The luminescence tracking properties of Eu3+-doped (red emission) and Tb3+-doped (green emission) NPs were successfully characterized by co-encapsulating with genetically modified cancer cell lines expressing green or red fluorescent proteins (GFP and RFP) in addition to a mixed population of live and dead cells stained with ethidium homodimer. Detailed quantification of the luminescent and fluorescent signals was performed to confirm no overlap between each of the NPs and between NPs and cells. Thus, the spectral independence of Eu3+-doped and Tb3+-doped NPs with each other and with common fluorophores highlights the potential application of this novel technique in multiplexed systems, where many such luminescent NPs (other doped and co-doped NPs) can be used to simultaneously track different input conditions on the same platform. Graphical abstract ᅟ.

Project description:Most individuals acutely infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit mild symptoms. However, 10 to 20% of those infected develop long-term symptoms, referred to as post-coronavirus disease 2019 (COVID-19) condition (PCC). One hypothesis is that PCC might be exacerbated by viral persistence in tissue sanctuaries. Therefore, the accurate detection and quantification of SARS-CoV-2 are not only necessary for viral load monitoring but also crucial for detecting long-term viral persistence and determining whether viral replication is occurring in tissue reservoirs. In this study, the sensitivity and robustness of reverse transcription (RT)-droplet digital PCR (ddPCR) and RT-quantitative PCR (qPCR) techniques have been compared for the detection and quantification of SARS-CoV-2 genomic and subgenomic RNAs from oropharyngeal swabs taken from confirmed SARS-CoV-2-positive, SARS-CoV-2-exposed, and nonexposed individuals as well as from samples from mice infected with SARS-CoV-2. Our data demonstrated that both techniques presented equivalent results in the mid- and high-viral-load ranges. Additionally, RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, allowing the accurate detection of positive results in individuals exposed to the virus. Overall, these data suggest that RT-ddPCR might be an alternative to RT-qPCR for detecting low viral loads in samples and for assessing viral persistence in samples from individuals with PCC. IMPORTANCE We developed one-step reverse transcription (RT)-droplet digital PCR (ddPCR) protocols to detect SARS-CoV-2 RNA and compared them to the gold-standard RT-quantitative PCR (RT-qPCR) method. RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, while both techniques were equivalent in the mid- and high-viral-load ranges. Overall, these results suggest that RT-ddPCR might be a viable alternative to RT-qPCR when it comes to detecting low viral loads in samples, which is a highly relevant issue for determining viral persistence in as-yet-unknown tissue reservoirs in individuals suffering from post-COVID conditions or long COVID.

Dataset Information

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

Publications

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

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