Abstract
Digital bioassays, popularized by digital PCR, provide some of the most robust and accurate methods for nucleic acid quantification. In this chapter, we detail a protocol for digital, isothermal, and multiplex detection of microRNAs, which relies on a recently developed amplification method. Our approach uses programmable ultrasensitive molecular amplifiers (PUMAs) to reveal the presence of target microRNAs randomly isolated in picoliter-size microfluidic droplets. Nonspecific amplification in droplets that do not contain a target is eliminated by an active threshold mechanism. Multiple circuits can be assembled for the multiplex digital detection of up to three targets. We finally present the option of using fluorescent dropcodes to streamline the assay and analyze more than a dozen samples in parallel.
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Acknowledgments
Our research is supported by grants from the University Paris Sciences et Lettres and the European Research Council (ERC-2020-STG MoP-MiP 939394).
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Rondelez, Y., Gines, G. (2023). Programmable Ultrasensitive Molecular Amplifier for Digital and Multiplex MicroRNA Quantification. In: Dalmay, T. (eds) MicroRNA Detection and Target Identification. Methods in Molecular Biology, vol 2630. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2982-6_7
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DOI: https://doi.org/10.1007/978-1-0716-2982-6_7
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