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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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Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in genome-wide studies to compare gene or protein expression under two varied conditions, such as healthy and diseased states. Microarrays consist of glass or silica slides on which probe molecules are covalently attached through surface functionalization. Most commonly, the slides are prepared through the chemisorption of silanes to silica...

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Related Experiment Video

Updated: Jul 6, 2025

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Published on: August 10, 2017

Quantitative mRNA expression measurement at home.

Sonalisa Pandey^1,2, Sara Safa McCoy^2,3, Tsering Stobdan²

¹Shanvi, San Diego, CA, USA.

Scientific Reports

|January 10, 2024

Summary

This summary is machine-generated.

This study introduces a rapid, fluorescence-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for accurate mRNA measurement. This accessible technique offers a viable alternative to expensive RT-PCR, enabling point-of-care diagnostics.

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Last Updated: Jul 6, 2025

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Published on: February 25, 2017

Area of Science:

Molecular Biology
Biotechnology
Diagnostics

Background:

Reverse transcription polymerase chain reaction (RT-PCR) is the standard for mRNA measurement but requires specialized equipment and expertise.
Loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detection method, but its sensitivity for RNA detection has historically lagged behind RT-PCR.
Accurate and accessible mRNA quantification is crucial for various biological and clinical applications.

Purpose of the Study:

To develop and validate a fluorescence-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) protocol for sensitive and accurate mRNA quantification.
To compare the performance of the developed RT-LAMP assay with established RT-PCR methods.
To demonstrate the utility of the RT-LAMP assay for diverse sample types and its potential for point-of-care applications.

Main Methods:

A novel fluorescence-based RT-LAMP protocol was developed for measuring CDX2 mRNA expression.
A new primer design strategy was implemented to minimize false positives and enhance quantification accuracy.
The assay was validated using various sample types, including cDNA, mRNA, and direct tissue samples.

Main Results:

The developed RT-LAMP protocol demonstrated high correlation with RT-PCR standards for CDX2 expression (r=0.99, p<0.001).
The assay provided accurate quantification across diverse sample types, including direct tissue samples.
The entire measurement process, from sample to result, was completed in approximately 25 minutes, significantly faster than RT-PCR (over 3 hours).

Conclusions:

Fluorescence-based RT-LAMP offers a sensitive, accurate, and rapid alternative to RT-PCR for mRNA measurement.
The developed protocol and primer design strategy overcome previous limitations of LAMP sensitivity and specificity.
The assay's simplicity and speed, coupled with a portable device, facilitate at-home, outdoor, and point-of-care mRNA expression analysis.