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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: Nov 5, 2025

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Published on: March 31, 2021

Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control.

Aileen G Rowan¹, Philippa May², Anjna Badhan³

¹Section of Virology, Department of Infectious Disease, Imperial College London, United Kingdom; Centre for Haematology, Department of Infection and Inflammation, Imperial College London, United Kingdom.

Journal of Virological Methods

|May 13, 2021

Summary

Quantifying SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) viral load is feasible in clinical settings. An internal control identified inadequate samples, preventing false negatives and enabling viral burden-guided decisions for COVID-19 management.

Keywords:

COVID-19 RNase P RT-qPCR SARS-CoV-2 Viral burden

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Quantification and Whole Genome Characterization of SARS-CoV-2 RNA in Wastewater and Air Samples

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Last Updated: Nov 5, 2025

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

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Published on: March 31, 2021

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

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Quantification and Whole Genome Characterization of SARS-CoV-2 RNA in Wastewater and Air Samples

Quantification and Whole Genome Characterization of SARS-CoV-2 RNA in Wastewater and Air Samples

Published on: June 30, 2023

Area of Science:

Virology
Molecular Biology
Clinical Diagnostics

Background:

Accurate measurement of SARS-CoV-2 viral load is crucial for managing COVID-19 patients and carriers.
Clinical settings require reliable methods for SARS-CoV-2 quantification to inform patient care and public health strategies.

Purpose of the Study:

To establish a feasible clinical assay for quantifying SARS-CoV-2 viral copy number.
To implement an internal control for assessing sample quality and assay reliability.

Main Methods:

Development of a duplex RT-qPCR assay targeting the SARS-CoV-2 E gene and the human RNase P gene.
Utilizing RNase P amplification as an internal control for sample sufficiency and integrity.
Establishing a standard curve for converting Cq values to viral copy numbers.

Main Results:

The duplex RT-qPCR assay is clinically feasible and highly reproducible, linear across 7 logs.
An internal control identified 2.4% of nasopharyngeal swabs as inadequate for testing.
SARS-CoV-2 copy number was independent of RNase P copy number, ensuring comparability between samples.

Conclusions:

Quantification of SARS-CoV-2 viral load is achievable in a clinical setting.
The assay's internal control is vital for detecting inadequate samples and preventing false negatives.
Accurate viral load quantification facilitates evidence-based clinical and public health decision-making for COVID-19.