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Real Time RT-PCR

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The real-time quantification of the number of amplified products is...

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Related Experiment Video

Updated: Aug 13, 2025

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Published on: August 3, 2011

Programmable Ultrasensitive Molecular Amplifier for Digital and Multiplex MicroRNA Quantification.

Yannick Rondelez¹, Guillaume Gines²

¹Gulliver Laboratory, ESPCI Paris - Université PSL, Paris, France.

Methods in Molecular Biology (Clifton, N.J.)

|January 23, 2023

Summary

This study introduces programmable ultrasensitive molecular amplifiers (PUMAs) for accurate digital detection of microRNAs. This novel method enables sensitive, multiplexed quantification in microfluidic droplets with an active threshold mechanism.

Keywords:

DNA circuit Digital assay Droplet microfluidics MicroRNA Molecular programming Multiplexing PUMA

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Last Updated: Aug 13, 2025

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Published on: August 3, 2011

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

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Published on: April 14, 2015

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Published on: June 26, 2016

Area of Science:

Molecular Biology
Biotechnology
Analytical Chemistry

Background:

Digital bioassays, including digital PCR, offer high accuracy in nucleic acid quantification.
MicroRNA detection is crucial for understanding biological processes and disease states.

Purpose of the Study:

To detail a protocol for digital, isothermal, and multiplex detection of microRNAs using a novel amplification method.
To demonstrate the capability of programmable ultrasensitive molecular amplifiers (PUMAs) for sensitive microRNA quantification.

Main Methods:

Utilizing programmable ultrasensitive molecular amplifiers (PUMAs) for amplification of target microRNAs.
Employing picoliter-sized microfluidic droplets for random isolation of nucleic acids.
Implementing an active threshold mechanism to eliminate nonspecific amplification.
Assembling multiple circuits for multiplex digital detection of up to three targets.
Optionally using fluorescent dropcodes for streamlined assay analysis and parallel sample processing.

Main Results:

Successful digital, isothermal, and multiplex detection of microRNAs was achieved.
The PUMA method demonstrated high sensitivity in detecting target microRNAs within droplets.
The active threshold mechanism effectively prevented nonspecific amplification.
Multiplex detection of up to three microRNA targets was feasible.

Conclusions:

The developed PUMA-based digital bioassay provides a robust and accurate method for microRNA quantification.
This approach offers sensitive, multiplexed, and specific detection, overcoming limitations of traditional methods.
The integration of fluorescent dropcodes enhances throughput and streamlines sample analysis.