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Real Time RT-PCR

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: Feb 22, 2026

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

Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification.

Peter Androvic^1,2, Lukas Valihrach¹, Julie Elling³

¹Laboratory of Gene Expression, Institute of Biotechnology CAS, Biocev, Vestec 252 50, Czech Republic.

Nucleic Acids Research

|September 16, 2017

Summary

We developed Two-tailed RT-qPCR, a sensitive and cost-effective method for quantifying microRNA (miRNA) expression. This novel system accurately measures miRNA levels in biological samples, offering biomarker potential.

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Last Updated: Feb 22, 2026

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

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

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Published on: February 12, 2018

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Published on: November 18, 2014

Area of Science:

Molecular Biology
Biochemistry
Genetics

Background:

MicroRNAs (miRNAs) are key posttranscriptional gene regulators.
Their stability in body fluids makes them promising biomarkers.
Accurate quantification of miRNA expression is crucial for research and diagnostics.

Purpose of the Study:

To develop a highly specific, sensitive, and cost-effective system for miRNA quantification.
To introduce a novel method, Two-tailed RT-qPCR, for precise miRNA expression analysis.
To enable the detection of the full isomiR repertoire for comprehensive miRNA profiling.

Main Methods:

Developed Two-tailed RT-qPCR using novel target-specific primers with two hemiprobes and a hairpin structure.
Employed SYBR-green detection chemistry for two-step RT-qPCR.
Validated the method's sensitivity, specificity, and dynamic range.

Main Results:

Two-tailed RT-qPCR demonstrated high specificity and sensitivity, detecting down to ten target miRNA molecules.
The method accurately captured the complete miRNA isomiR repertoire.
Achieved excellent correlation (r² = 0.985) with standard TaqMan miRNA assays.
Total analysis time was reduced to less than 2.5 hours.

Conclusions:

Two-tailed RT-qPCR provides a robust, rapid, and cost-effective solution for miRNA expression profiling.
The system's high sensitivity and specificity facilitate accurate miRNA biomarker discovery.
This method enhances the comprehensive analysis of miRNA content in biological samples.