title: Ultrahigh-throughput Mammalian single-cell reverse-transcriptase polymerase chain reaction in microfluidic drops.
abstract: The behaviors of complex biological systems are often dictated by the properties of their heterogeneous and sometimes rare cellular constituents. Correspondingly, the analysis of individual cells from a heterogeneous population can reveal information not obtainable by ensemble measurements. Reverse-transcriptase polymerase chain reaction (RT-PCR) is a widely used method that enables transcriptional profiling and sequencing analysis on bulk populations of cells. Major barriers to successfully implementing this technique for mammalian single-cell studies are the labor, cost, and low-throughput associated with current approaches. In this report, we describe a novel droplet-based microfluidic system for performing ~50000 single-cell RT-PCR reactions in a single experiment while consuming a minimal amount of reagent. Using cell type-specific staining and TaqMan RT-PCR probes, we demonstrate the identification of specific cells from a mixed human cell population. The throughput, robust detection rate and specificity of this method makes it well-suited for characterizing large, heterogeneous populations of cells at the transcriptional level.
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, California 94158, United States.
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Eastburn
authors.forename: Adam
authors.initials: A
authors.lastname: Sciambi
authors.forename: Adam R
authors.initials: AR
authors.lastname: Abate
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D017931:DNA Primers; D057166:High-Throughput Screening Assays; D006801:Humans; D008297:Male; D044085:Microfluidics / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers