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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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Updated: May 28, 2026

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

Published on: April 5, 2018

False negative results from using common PCR reagents.

Dean J Bacich¹, Kathryn M Sobek, Jessica L Cummings

¹Department of Urology, University of Pittsburgh, 5200 Centre Avenue, Pittsburgh, PA 15232, USA. okeefeds@upmc.edu.

BMC Research Notes

|October 29, 2011

Summary

Uracil-DNA-glycosylase (UNG) prevents PCR contamination but can cause false negatives. Minute amounts of UNG-treated PCR products or primer-dimers can inhibit amplification of legitimate DNA targets.

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Last Updated: May 28, 2026

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

Published on: April 5, 2018

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

Published on: June 20, 2018

Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

Polymerase Chain Reaction (PCR) is sensitive for detecting novel viruses.
Carry-over contamination is a known issue in PCR.
Uracil-DNA-glycosylase (UNG) is commonly used in PCR master mixes to prevent contamination.

Purpose of the Study:

To investigate the impact of UNG on PCR sensitivity.
To identify potential causes of false-negative PCR results.

Main Methods:

Testing PCR assay sensitivity for murine DNA contamination.
Evaluating the effect of UNG-digested PCR products and primer-dimers on amplification.

Main Results:

Minute quantities of UNG-digested PCR product can inhibit amplification.
Primer-dimers in negative controls can also block legitimate DNA amplification.
UNG presence or absence did not alter the inhibitory effect of these contaminants.

Conclusions:

UNG, while preventing contamination, may lead to false-negative PCR results.
This phenomenon could explain discrepant findings in studies of MLV-related viruses.
Careful control of potential false negatives is crucial in low-target-copy-number PCR experiments.