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Highly multiplexed single-cell quantitative PCR.

ABSTRACT: We present a microfluidic device for rapid gene expression profiling in single cells using multiplexed quantitative polymerase chain reaction (qPCR). This device integrates all processing steps, including cell isolation and lysis, complementary DNA synthesis, pre-amplification, sample splitting, and measurement in twenty separate qPCR reactions. Each of these steps is performed in parallel on up to 200 single cells per run. Experiments performed on dilutions of purified RNA establish assay linearity over a dynamic range of at least 104, a qPCR precision of 15%, and detection sensitivity down to a single cDNA molecule. We demonstrate the application of our device for rapid profiling of microRNA expression in single cells. Measurements performed on a panel of twenty miRNAs in two types of cells revealed clear cell-to-cell heterogeneity, with evidence of spontaneous differentiation manifested as distinct expression signatures. Highly multiplexed microfluidic RT-qPCR fills a gap in current capabilities for single-cell analysis, providing a rapid and cost-effective approach for profiling panels of marker genes, thereby complementing single-cell genomics methods that are best suited for global analysis and discovery. We expect this approach to enable new studies requiring fast, cost-effective, and precise measurements across hundreds of single cells.

SUBMITTER: VanInsberghe M

PROVIDER: S-EPMC5788347 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Highly multiplexed single-cell quantitative PCR.

VanInsberghe Michael M Zahn Hans H White Adam K AK Petriv Oleh I OI Hansen Carl L CL

PloS one 20180129 1

We present a microfluidic device for rapid gene expression profiling in single cells using multiplexed quantitative polymerase chain reaction (qPCR). This device integrates all processing steps, including cell isolation and lysis, complementary DNA synthesis, pre-amplification, sample splitting, and measurement in twenty separate qPCR reactions. Each of these steps is performed in parallel on up to 200 single cells per run. Experiments performed on dilutions of purified RNA establish assay linea ...[more]

PMID: 29377915

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Project description:Restenosis, re-narrowing of arterial lumen following intervention for cardiovascular disease, remains a major issue limiting the long-term therapeutic efficacy of treatment. The signaling molecules, TGFβ (transforming growth factor-beta) and Smad3, play important roles in vascular restenosis, but very little is yet known about the down-stream dynamics in global protein expression and phosphorylation. Here, we develop a highly multiplexed quantitative proteomic and phosphoproteomic strategy employing 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags and The DiLeu Tool software to globally assess protein expression and phosphorylation changes in smooth muscle cells (SMCs) treated with TGFβ/Smad3 and/or SDF-1α (stromal cell-derived factor). A total of 4086 proteins were quantified in the combined dataset of proteome and phosphoproteome across 12-plex DiLeu-labeled SMC samples. 2317 localized phosphorylation sites were quantified, corresponding to 1193 phosphoproteins. TGFβ/Smad3 induced up-regulation of 40 phosphosites and down-regulation of 50 phosphosites, and TGFβ/Smad3-specific SDF-1α exclusively facilitated up-regulation of 27 phosphosites and down-regulation of 47 phosphosites. TGFβ/Smad3 inhibited the expression of contractile-associated proteins including smooth muscle myosin heavy chain, calponin, cardiac muscle alpha-actin, and smooth muscle protein 22α. Gene ontology and pathway enrichment analysis revealed that elevated TGFβ/Smad3 activated cell proliferation and TGFβ signaling pathway, sequentially stimulating phosphorylation of CXCR4 (C-X-C chemokine receptor 4). SDF-1α/CXCR4 activated extracellular signal-regulating kinase signaling pathway and facilitated the expression of synthetic marker, osteopontin, which was validated through targeted analysis. These findings provide new insights into the mechanisms of TGFβ regulated SMC dedifferentiation, as well as new avenues for designing effective therapeutics for vascular disease.

Dataset Information

Highly multiplexed single-cell quantitative PCR.

Publications

Highly multiplexed single-cell quantitative PCR.

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