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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 miRNA in two types of cells revealed clear cell-to-cell heterogeneity, with evidence of spontaneous differentiation manifest 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.

OTHER RELATED OMICS DATASETS IN: PRJNA398516

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE102734 | GEO | 2018/01/31

REPOSITORIES: GEO

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Project description:Tissues are composed of highly heterogeneous mixtures of cell subtypes, and this diversity is increasingly being characterized using high-throughput single cell analysis methods. However, these efforts are hindered by the fact that tissues must first be dissociated into single cell suspensions that are viable and still accurately represent phenotypes from the original tissue. Current methods for breaking down tissues are inefficient, labor-intensive, subject to high variability, and potentially biased towards cell subtypes that are easier to release. Here, we present a microfluidic platform consisting of three different tissue processing technologies that can perform the complete tissue to single cell workflow, including digestion, disaggregation, and filtration. First, we developed a new microfluidic digestion device that can be loaded with minced tissue specimens quickly and easily, and then use the combination of proteolytic enzyme activity and fluid shear forces to accelerate tissue breakdown. Next, we integrated dissociation and filter technologies into a single device, which enhanced single cell numbers and fully prepared the sample for single cell analysis. The final multi-device platform was then evaluated using a diverse array of tissue types that exhibited a wide range of properties. For murine kidney and mammary tumor, we found that microfluidic processing produced 2.5-fold more single, viable cells. Single cell RNA sequencing (scRNA-seq) further revealed that device processing enriched for endothelial cells, fibroblasts, and basal epithelium, and did not increase stress responses. For murine liver and heart, which are softer tissues containing fragile cell types, processing time could be reduced to 15 min, and even as short as 1 min. We also demonstrated that periodic recovery at defined time intervals produced substantially more hepatocytes and cardiomyocytes than continuous operation, most likely by preventing damage to fragile cell types. In future work, we will seek to integrate additional operations such as upstream tissue preparation and downstream microfluidic cell sorting and detection to create powerful point-of-care single cell diagnostic platforms.

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Highly multiplexed single-cell quantitative PCR

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