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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: Nov 18, 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

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.

Thomas G W Graham¹, Claire Dugast-Darzacq¹, Gina M Dailey¹

¹Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, United States of America.

|February 3, 2021

Summary

This summary is machine-generated.

Simple, inexpensive RNA extraction and RT-qPCR methods for COVID-19 testing are evaluated. These open-source protocols, including direct swab addition and end-point fluorescence, show potential to reduce testing time and cost.

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Last Updated: Nov 18, 2025

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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:

Molecular Biology
Virology
Public Health

Background:

Community reopening during the COVID-19 pandemic necessitates widespread SARS-CoV-2 testing.
Current testing methods can be time-consuming and expensive, hindering rapid deployment.
Development of simplified, cost-effective diagnostic protocols is crucial for pandemic management.

Purpose of the Study:

To evaluate simpler and less expensive RNA extraction and RT-qPCR protocols for SARS-CoV-2 detection.
To validate open-source methods for improved accessibility and reduced cost in COVID-19 testing.
To explore alternative diagnostic readouts for RT-qPCR to bypass specialized equipment.

Main Methods:

Isopropanol precipitation for RNA extraction from nasopharyngeal swabs.
Direct addition of swab samples to RT-qPCR without a separate RNA extraction step, using a validated collection solution.
Development and validation of an open-source RT-qPCR master mix.
Evaluation of end-point fluorescence imaging as a diagnostic readout.

Main Results:

Isopropanol precipitation effectively extracts RNA from nasopharyngeal swabs.
Direct swab addition to RT-qPCR is feasible with a suitable collection solution.
The open-source master mix detected viral RNA with a limit of detection around 50 RNA copies/reaction.
End-point fluorescence imaging provided accurate diagnostic results without a thermocycler.
Homemade master mix showed strong correlation (r2=0.98) with commercial kits for clinical samples.

Conclusions:

Simplified RNA extraction and RT-qPCR protocols can be effective for SARS-CoV-2 detection.
Open-source methods, including direct swab addition and end-point fluorescence, offer potential for reduced cost and faster turnaround times for COVID-19 testing.
These accessible methods could significantly aid global efforts in widespread SARS-CoV-2 surveillance and diagnosis.