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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: May 22, 2025

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Published on: August 3, 2011

Volumetric, Microfluidic Plasmonic RT-PCR.

Harshit Harpaldas Chellani¹, Kelia Human¹, Robert Stanciu¹

¹Department of Biomedical Engineering, Columbia University, New York, NY, 10027, USA.

|March 12, 2025

Summary

This summary is machine-generated.

This study introduces a rapid, microfluidic plasmonic reverse transcription (RT)-PCR method for decentralized pathogen detection. It achieves SARS-CoV-2 amplification in just 16 minutes using light-activated nanoparticles and closed-loop temperature control.

Keywords:

microfluidics pathogen detection plasmonic PCR point‐of‐care diagnostics

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Last Updated: May 22, 2025

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Area of Science:

Biotechnology
Nanotechnology
Molecular Diagnostics

Background:

Decentralized molecular detection of pathogens is crucial for public health.
Traditional polymerase chain reaction (PCR) thermocycling faces challenges in point-of-care settings.
Plasmonic PCR, utilizing light-activated nanomaterials, offers a promising alternative for rapid thermocycling.

Purpose of the Study:

To develop and demonstrate a volumetric, microfluidic plasmonic reverse transcription (RT)-PCR method.
To integrate closed-loop temperature control for enhanced precision and speed.
To enable rapid, compact, and decentralized pathogen detection.

Main Methods:

Fabrication of a microfluidic chip with an integrated thermocouple for real-time temperature monitoring.
Implementation of a proportional-integral-derivative (PID) algorithm for closed-loop temperature control using an infrared LED.
Dispersion of gold nanorods in solution with RT-PCR reagents for light-induced heating.
Development of an instrument incorporating an infrared LED, fan, and fluorometer for plasmonic RT-PCR and detection.

Main Results:

Achieved rapid thermocycling and amplification of SARS-CoV-2 in 16 minutes (5 min RT, 11 min for 45 cycles).
Demonstrated volumetric plasmonic PCR within a microfluidic chip with precise temperature control.
Validated the effectiveness of integrated thermocouples and PID control for rapid thermocycling.

Conclusions:

The developed microfluidic plasmonic RT-PCR system enables significantly faster pathogen detection.
This approach holds promise for rapid, compact, and decentralized molecular diagnostics.
Integration of microfluidics and nanomaterials is key to advancing point-of-care molecular testing.