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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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Author Spotlight: Optimizing Digital Droplet PCR Method for Accurate Adeno-Associated Viral Genome Quantification

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Quantifying Rhinovirus Genomic and Negative-Strand RNA by RT-PCR.

Shahina Wiehler¹, Michelle E Love¹, David Proud²

¹Department of Physiology and Pharmacology, and Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.

Methods in Molecular Biology (Clifton, N.J.)

|February 27, 2025

Summary

This summary is machine-generated.

Quantitative RT-PCR assays for rhinovirus RNA were developed. These methods measure both genomic and negative-strand RNA, aiding in understanding viral replication and immune response activation.

Keywords:

Airway epithelium Human rhinovirus Negative-strand RNA Real-time PCR genomic RNA

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Area of Science:

Virology
Molecular Biology
Immunology

Background:

Rhinovirus replication involves generating viral genomic RNA and a negative-strand template.
Negative-strand RNA assays can quantify double-stranded RNA, which activates host immune responses via pattern recognition receptors.

Purpose of the Study:

To provide quantitative RT-PCR (reverse transcription-polymerase chain reaction) protocols for rhinovirus RNA.
To enable measurement of both rhinovirus genomic RNA and negative-strand RNA.

Main Methods:

Development and validation of RT-PCR protocols.
Quantitative measurement of viral genomic RNA.
Quantitative measurement of negative-strand rhinovirus RNA.

Main Results:

Established RT-PCR assays for accurate quantification of rhinovirus genomic RNA.
Established RT-PCR assays for accurate quantification of negative-strand rhinovirus RNA.
Demonstrated the utility of negative-strand RNA assays for assessing viral replication levels.

Conclusions:

The developed RT-PCR protocols offer reliable methods for quantifying rhinovirus RNA species.
These assays facilitate research into rhinovirus replication dynamics and host-pathogen interactions.
Understanding double-stranded RNA levels is crucial for investigating rhinovirus-induced immune responses.