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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Related Experiment Video

Updated: Jun 21, 2026

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays
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Accurate and Microfluidics-Free Digital Nucleic Acid Quantification with Crescent Microbeads-Templated Emulsions.

Long Chen1, Yanan Du1, Sixiang Rao1

  • 1School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China.

ACS Sensors
|July 12, 2025
PubMed
Summary

A new microfluidics-free droplet digital PCR method uses crescent microbeads for accurate nucleic acid quantification. This innovation enhances accessibility and performance, overcoming limitations of current droplet digital PCR systems.

Keywords:
crescent microbeads, dropletdigital polymerase chain reaction (dPCR)nucleic acidpipetting

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Analytical Chemistry

Background:

  • Droplet digital PCR (ddPCR) offers absolute nucleic acid quantification without standard curves.
  • Current ddPCR methods often require complex microfluidics and skilled personnel.
  • Existing microfluidics-free methods struggle with limited volumes and inconsistent droplet sizes.

Purpose of the Study:

  • To develop a microfluidics-free ddPCR strategy for accurate nucleic acid quantification.
  • To address limitations of existing ddPCR technologies, particularly in resource-constrained settings.
  • To improve accessibility and applicability of ddPCR for diagnostics.

Main Methods:

  • Utilized crescent microbeads generated via aqueous two-phase system (ATPS) for uniform droplet generation.
  • Optimized surfactant types, pipetting cycles, and washing procedures for droplet quality.
  • Employed a pipetting-based method for microfluidics-free droplet generation and nucleic acid quantification.

Main Results:

  • Achieved high size uniformity of crescent microbeads and consistent droplet generation.
  • Demonstrated precise quantification of DNA gradient dilutions, comparable to commercial ddPCR systems.
  • Showcased a 57.3% improvement in positive ratio quantification compared to spherical microbeads.
  • Successfully quantified synthetic KRAS gene mutations and clinical HPV samples.

Conclusions:

  • The crescent microbead-based strategy provides accurate and accessible ddPCR.
  • This method overcomes technological and financial barriers in nucleic acid diagnostics.
  • Enhances ddPCR applicability in resource-limited environments, potentially transforming diagnostics.