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Related Experiment Video

Updated: Nov 3, 2025

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides
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Electrophoretic mobility shift as a molecular beacon-based readout for miRNA detection.

Getulio P Oliveira-Jr1, Raquel H Barbosa1, Lauren Thompson2

  • 1Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States.

Biosensors & Bioelectronics
|June 1, 2021
PubMed
Summary

This study introduces a novel, rapid method for quantifying microRNAs (miRNAs) using molecular beacons and delayed electrophoresis. This technique offers sensitive and specific detection of miRNA hybridization, improving upon existing laborious methods.

Keywords:
Electrophoretic mobilityGel electrophoresisMolecular beaconsmicroRNA (miRNA)

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MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as A Novel Detection and Quantification Method

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • MicroRNAs (miRNAs) are key regulators of gene expression.
  • Current miRNA quantification methods (Northern blotting, PCR) are time-consuming and labor-intensive.
  • Molecular beacons (MBs) offer rapid miRNA detection but require complex readout systems.

Purpose of the Study:

  • To develop a novel, rapid, and affordable method for quantitative miRNA detection.
  • To bypass the need for sophisticated readout methods in miRNA quantification.
  • To validate miRNA-target binding through orthogonal detection.

Main Methods:

  • Utilized molecular beacons (MBs) for hybridization with target miRNAs.
  • Employed delayed electrophoretic mobility to detect miRNA-MB hybridization.
  • MBs form fluorescent duplexes with reduced electrophoretic mobility upon hybridization.

Main Results:

  • The method provides quantitative detection based on altered electrophoretic mobility.
  • Fluorescent band location on the gel confirms target identity, ensuring specificity.
  • Achieved a limit of detection of approximately 100 pM with single-nucleotide specificity.
  • Successfully detected specific red blood cell miRNAs in total RNA samples.

Conclusions:

  • A rapid, affordable, and sensitive method for detecting single-stranded DNA and RNA sequences, including miRNAs, has been developed.
  • Delayed electrophoretic mobility offers a simplified readout for molecular beacon-based detection.
  • The method enhances miRNA detection capabilities for research and clinical applications.