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A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells
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Sensitively distinguishing intracellular precursor and mature microRNA abundance

Fan Yang1,2, Yaru Cheng1,2, Yu Cao1,2

  • 1Beijing Advanced Innovation Center for Materials Genome Engineering , University of Science and Technology Beijing , 30 Xueyuan Road , Beijing 100083 , P. R. China . Email: hfdong@ustb.edu.cn ;

Chemical Science
|March 8, 2019
PubMed

Insights

This study introduces a new method using DNA probes to detect and differentiate between microRNAs (miRNAs) and precursor microRNAs (pre-miRNAs) in living cells. This advance aids in understanding miRNA roles in diseases like cancer.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biomedical Diagnostics

Background:

  • MicroRNAs (miRNAs) and precursor microRNAs (pre-miRNAs) are crucial regulators in cellular processes and disease.
  • Distinguishing between mature miRNAs and pre-miRNAs is challenging due to sequence homology, hindering accurate diagnosis and prognosis.
  • Existing detection methods struggle with low-abundance targets and differentiating similar sequences.

Purpose of the Study:

  • To develop a novel method for sensitive and discriminatory detection of miRNAs versus pre-miRNAs in living cells.
  • To establish a tool for assessing intracellular miRNA and pre-miRNA expression levels.
  • To advance the biomedical applications of miRNA research.

Main Methods:

  • Utilized a novel cascade reaction involving two pairs of programmable hairpin oligonucleotide probes.
  • Employed a specific hybridization chain reaction (HCR) triggered by target miRNAs or pre-miRNAs.
  • Developed a system for homogeneous solution-based detection with fluorescence readout.

Main Results:

  • Achieved sensitive and specific discrimination between miRNAs and pre-miRNAs.
  • Demonstrated successful assessment of intracellular miRNA and pre-miRNA abundance in various living cells.
  • Observed a significant increase in fluorescence intensity upon successful target recognition and HCR initiation.

Conclusions:

  • The developed cascade reaction system provides a novel tool for accurate and discriminatory assessment of miRNA and pre-miRNA abundance.
  • This method enables real-time monitoring of miRNA and pre-miRNA expression in living cells.
  • The findings support potential biomedical applications in cancer diagnosis and prognosis.

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