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This low cost, sensitive, and adaptable technique will provide an accessible platform for high throughput single-cell analysis and enable a wide range of research and clinical applications. Using mixtures of in vitro transcripts and murine cell lines, we demonstrated the detection of single RNA molecules and rare cell populations at a frequency of 0.1%. We also developed a simulation model to guide experimental design and optimize the accuracy and precision of the assay. Fluorescent signals indicative of gene expressions are analyzed using a probabilistic deconvolution method to account for ambient RNA and cell doublets and produce single-cell gene signature profiles, as well as predict cell frequencies within heterogeneous samples. Our customized reagent cocktail incorporates the bacteriophage T7 gene 2.5 protein to overcome cell lysate-mediated inhibition and allows for one-step RT-PCR of single cells encapsulated in nanoliter droplets. Here, we present a novel microdroplet-based, one-step reverse-transcriptase polymerase chain reaction (RT-PCR) platform and demonstrate the detection of three targets simultaneously in over 100,000 single cells in a single experiment with a rapid read-out. Gene expression analysis of individual cells enables characterization of heterogeneous and rare cell populations, yet widespread implementation of existing single-cell gene analysis techniques has been hindered due to limitations in scale, ease, and cost.