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

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

Published on: April 14, 2015

Reverse transcription-based loop-mediated isothermal amplification strategy for real-time miRNA detection with

Abdu Ahmed Abdullah Al-Maskri¹, Jiawei Ye¹, Jadera Talap¹

¹Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Science, Zhejiang University, Hangzhou, Zhejiang, 310058, China.

Analytica Chimica Acta

|August 2, 2020

Abstract:

A novel reverse transcription-based loop-mediated isothermal amplification (LAMP) strategy for miRNA detection has been developed. This method consists of two stem-loop probes inspired by the dumbbell-shaped amplicons and inner primers used in conventional LAMP reactions. Termed "terminal hairpin formation and self-priming" (THSP), this reaction incorporates phosphorothioated (PS) modifications to achieve DNA folding and extension without primers. The final signal is monitored by a sequence-specific detection probe, which minimizes the background noise. We suggest that our rapid, facile, and reliable LAMP method will be a promising candidate for detecting miRNA in biomedical applications.

Keywords:

Loop-mediated isothermal amplification Phosphorothioated modifications Self-priming Terminal hairpin miRNA detection

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Real Time RT-PCR

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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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