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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: Jul 8, 2026

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Published on: April 9, 2014

Novel FRET-based assay to detect reverse transcriptase activity using modified dUTP analogues.

Joseph F Krebs¹, Anilkumar R Kore

¹Ambion/Applied Biosystems, Research and Development Department, 2130 Woodward Austin, Texas 78744, USA. jkrebs@ambion.com

Bioconjugate Chemistry

|January 1, 2008

Summary

A new continuous assay measures reverse transcriptase (RT) polymerase activity using FRET. This method aids in characterizing RT enzymes and identifying potential antiviral drug candidates.

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Last Updated: Jul 8, 2026

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

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Published on: April 9, 2014

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

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Quantitative Structure-Activity Relationship, Activity Prediction, and Molecular Dynamics of Non-nucleotide Reverse Transcriptase Inhibitors

Quantitative Structure-Activity Relationship, Activity Prediction, and Molecular Dynamics of Non-nucleotide Reverse Transcriptase Inhibitors

Published on: May 9, 2025

Area of Science:

Biochemistry
Molecular Biology
Enzymology

Background:

Reverse transcriptase (RT) is crucial for viral replication.
Accurate measurement of RT polymerase activity is essential for drug discovery.

Purpose of the Study:

To develop a novel continuous assay for measuring reverse transcriptase (RT) polymerase activity.
To characterize RT enzyme thermostability and inhibitor effects.

Main Methods:

Utilized fluorescence energy transfer (FRET) measurements.
Employed fluorescently labeled deoxyuridine incorporation into cDNA.
Used polyA/oligo dT primer/template for assay validation.

Main Results:

The assay accurately detects viral RT polymerase activity.
Compared thermostability of AMV, HIV-1, and MMLV RT enzymes.
Demonstrated increased thermostability with primer/template presence.
Studied inhibitor effects on HIV-1 RT activity.

Conclusions:

The developed FRET-based assay is a sensitive tool for RT activity measurement.
The assay facilitates RT enzyme characterization and inhibitor screening.
This method can accelerate the development of novel antiviral therapeutics.