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. 2021 Mar 24;11:6777. doi: 10.1038/s41598-021-86087-4

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Simultaneous detection of three targets by our droplet RT-PCR platform at single molecule level is demonstrated using limiting dilutions of IVTs. (a) Samples containing various dilutions of IL-7Rα, Gata3, and EPCR IVTs were encapsulated for droplet RT-PCR analysis. A substantial increase in fluorescent signal was observed and quantified. The concentration of IVT in each sample was then estimated based on the fraction of droplets with significant increases in fluorescent signal and Poisson statistics. (b) Images of droplets containing a mixture of IL-7Rα, Gata3, and EPCR IVTs after amplification targeting the three genes. NTC No template control. (c) The distribution of fluorescence intensity of each droplet. The colours represent different populations clustered using DBSCAN, and were assigned based on the table in (A) to indicate the presence or absence of the three transcripts in each subpopulation. (d) A representative plot showing the correlation between input and estimated concentration of IL-7Rα IVTs using RT-qPCR and droplet RT-PCR. (e) The empirically determined dilution factors highly correlated with the sample dilution factors with both regression coefficient and correlation coefficient close to 1 (P < 0.01). Each dot represents one technical replicate. Data was analyzed and plotted using JMP (version 15.2.1 www.jmp.com).