A long-sought milestone in microfluidics research has been the development of integrated technology for scalable analysis of transcription in single cells. Here we present a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run. Our device executes all steps of single-cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR. In addition to higher throughput and reduced cost, we show that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specificity. We apply this technology to 3,300 single-cell measurements of ( i ) miRNA expression in K562 cells, ( ii ) coregulation of a miRNA and one of its target transcripts during differentiation in embryonic stem cells, and ( iii ) single nucleotide variant detection in primary lobular breast cancer cells. The core functionality established here provides the foundation from which a variety of on-chip single-cell transcription analyses will be developed.

Cells, even those having identical genotype, exhibit variability in their response to external stimuli. This variability arises from differences in the abundance, localization, and state of cellular components. Such nongenetic differences are likely heritable between successive generations and can also be influenced by processes such as cell cycle, age, or interplay between different pathways. To address the contribution of nongenetic heritability and cell cycle in cell-to-cell variability we developed a highthroughput and fully automated microfluidic platform that allows for concurrent measurement of gene expression, cell-cycle periods, age, and lineage information under a large number of temporally changing medium conditions and using multiple strains. We apply this technology to examine the role of nongenetic inheritance in cell heterogeneity of yeast pheromone signaling. Our data demonstrate that the capacity to respond to pheromone is passed across generations and that the strength of the response correlations between related cells is affected by perturbations in the signaling pathway. We observe that a ste50Δ mutant strain exhibits highly heterogeneous response to pheromone originating from a unique asymmetry between mother and daughter response. On the other hand, fus3Δ cells were found to exhibit an unusually high correlation between mother and daughter cells that arose from a combination of extended cell-cycle periods of fus3Δ mothers, and decreased cell-cycle modulation of the pheromone pathway. Our results contribute to the understanding of the origins of cell heterogeneity and demonstrate the importance of automated platforms that generate single-cell data on several parameters.single cell | microscopy | image analysis C ells must detect biochemical cues and respond appropriately to changing environmental conditions. However, under identical chemical stimuli not all cells respond the same. Heterogeneity in "cellular decision making" exists across all levels of life, from bacteria (1, 2) to simple eukaryotes (3-5) to mammalian cells (6, 7), and may be important in cellular adaptation to quickly changing microenvironments (8), in differentiation programs during development (6), and in sensitivity to drugs (9, 10). In addition to the intrinsic stochastic nature of gene expression (1, 3), each cell has an inherent capacity to respond, which is determined by the state and abundance of cellular components. This extrinsic variability may be passed across multiple generations, producing a nongenetic but heritable source of variation. Additional sources that contribute to extrinsic variability include asynchrony and interplay between different signaling pathways (11, 12), cell-cycle effects (3, 13), asymmetry in cellular divisions (14), and cellular aging (13).Investigating these effects requires tracking the genealogy of multiple generations of cells, followed by stimulation and quantitative single-cell measurement of response, including growth kinetics and gene expression. Although improved image analysis, ...

This work investigates the use of polydimethylglutarimide, or PMGI, as a structural material for surface micromachining. PMGI is a commercially available, positive-toned deep-UV resist designed for use in bi-layer lift-off techniques. This paper presents a technique for the microfabrication of free-standing PMGI structures, and uses those structures to extract the coefficient of thermal expansion and Young's modulus for PMGI. Our study found PMGI's coefficient of thermal expansion to be 56 ± 6 ppm • C −1 and Young's modulus to be 5.0 ± 0.5 GPa. Active structures were also fabricated by including a patterned metal layer. This allows the fabrication of active devices, such as bent-beam actuators. PMGI is a commercially available polymer being used in micromachining, and this paper provides the first report of its thermo-mechanical properties.

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