Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linearsignal across a wide range of total RNA concentrations and agreeswell with standard curve qPCR. The SD chip is found to digitize ahigh percentage (86.7%) of the sample for single-cell experiments.Moreover, quantification of transferrin receptor mRNA in single cellsagrees well with single-molecule fluorescence in situ hybridizationexperiments. The SD platform for absolute quantification of single-cellmRNA can be optimized for other genes and may be useful as an independentcontrol method for the validation of mRNA quantification techniques.
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