The study of single cells has evolved over the past several years to include expression and genomic analysis of an increasing number of single cells. Several studies have demonstrated wide spread variation and heterogeneity within cell populations of similar phenotype. While the characterization of these populations will likely set the foundation for our understanding of genomic- and expression-based diversity, it will not be able to link the functional differences of a single cell to its underlying genomic structure and activity. Currently, it is difficult to perturb single cells in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis. In order to address this challenge, scientists at Fluidigm developed a platform to integrate and miniaturize many of the experimental steps required to study single-cell function. The heart of this platform is an elastomer-based integrated fluidic circuit that uses fluidic logic to select and sequester specific single cells based on a phenotypic trait for downstream experimentation. Experiments with sequestered cells that have been performed include on-chip culture, exposure to various stimulants, and post-exposure image-based response analysis, followed by preparation of the mRNA transcriptome for massively parallel sequencing analysis. The flexible system embodies experimental design and execution that enable routine functional studies of single cells.
Total-RNA-based performance test (two-step)
workflow and pipetting map
(A) Workflow to prime Polaris IFC with beads, back-load them with RNAspikes 147, simulate front dosing with diluted ERCC spikes, and generate cDNA using mRNA-seq chemistry. The cDNA amplicons from Polaris IFC were analyzed on the Fluidigm M96.96 IFC using 85 qPCR assays for specific genes, 8 assays for ERCC RNA spikes, and 3 assays for RNAspikes 147. (B) Pipetting map for IFC prime step. During prime step, RNAspikes 147 are back-loaded to 8 specific inlets with cell capture beads. (C) Pipetting map for front-dosing simulation with ERCC RNA spikes, followed by mRNA-seq chemistry.