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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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Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing
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A Novel miRNA Detection Method Using Loop-Mediated Isothermal Amplification.

Saiwei Wu1, Abdu Ahmed Abdullah Al-Maskri2, Qun Li1

  • 1Department of Pharmacy, The Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu, Zhejiang, China.

International Journal of Analytical Chemistry
|September 21, 2023
PubMed
Summary

A new method uses ligation-based loop-mediated isothermal amplification (miRNA-LAMP) for rapid and ultrasensitive microRNA detection. This assay offers a sensitive quantitative analysis for clinical diagnosis and research.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Biochemistry

Background:

  • MicroRNAs (miRNAs) are crucial regulators of gene expression.
  • Sensitive and rapid detection methods for miRNAs are needed for clinical diagnosis and research.
  • Existing methods may lack the required sensitivity or speed for certain applications.

Purpose of the Study:

  • To develop a novel, rapid, and ultrasensitive method for microRNA detection.
  • To establish a ligation-based loop-mediated isothermal amplification (miRNA-LAMP) system.
  • To enable highly sensitive quantitative analysis of specific miRNAs.

Main Methods:

  • Designed two stem-loop DNA linker probes (A/B) that hybridize with the target miRNA.
  • Utilized SplintR Ligase for templated ligation of probes, forming a dumbbell-shaped amplicon.
  • Employed Bst 2.0 DNA polymerase and specific primers (BIP/FIP) for loop-mediated isothermal amplification (LAMP).
  • Incorporated EvaGreen fluorescent dye for real-time detection and quantification of amplification products.

Main Results:

  • Achieved a rapid and ultrasensitive miRNA detection system.
  • Demonstrated a linear detection range spanning seven orders of magnitude.
  • Established a low detection limit of 100 fM for the target miRNA (miR-200a).
  • Showcased greatly improved amplification efficiency through optimized LAMP conditions.

Conclusions:

  • The developed miRNA-LAMP assay is highly sensitive and quantitative.
  • This novel method provides a new avenue for precise miRNA analysis.
  • The assay offers significant convenience for clinical diagnostics and prognostic research.