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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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Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

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Published on: May 9, 2019

Protocol for qPCR analysis that corrects for cDNA amplification efficiency.

Mads V Damgaard¹, Jonas T Treebak¹

¹Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.

|July 12, 2022

Summary

This study introduces an improved quantitative polymerase chain reaction (qPCR) method. The enhanced protocol increases analytical accuracy by correcting for amplification efficiency and using multiple housekeeping genes, reducing statistical errors.

Keywords:

Gene Expression Molecular Biology

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Area of Science:

Molecular Biology
Biotechnology
Genetics

Background:

Quantitative polymerase chain reaction (qPCR) is a standard method for gene expression analysis.
The 2^-ΔΔCt technique is widely used but has limitations regarding amplification efficiency and the use of single housekeeping genes.
Variations in cDNA synthesis and PCR efficiency can impact the accuracy of qPCR results.

Purpose of the Study:

To present a modified qPCR protocol that addresses limitations of the standard 2^-ΔΔCt method.
To enhance the analytical accuracy of qPCR by accounting for variations in amplification efficiency.
To enable the use of multiple stable housekeeping genes for improved normalization.

Main Methods:

The protocol involves generating a standard curve on each qPCR plate.
It incorporates corrections for imperfect cDNA amplification efficiency.
The method allows for the normalization of gene expression data using multiple validated housekeeping genes.

Main Results:

The modified protocol demonstrates improved analytical accuracy compared to the standard 2^-ΔΔCt technique.
The approach effectively corrects for variations in cDNA amplification efficiency.
Utilizing multiple housekeeping genes enhances the reliability of expression data.

Conclusions:

This revised qPCR protocol offers a more robust and accurate method for gene expression analysis.
The protocol's ability to correct for amplification efficiency and use multiple housekeeping genes reduces statistical errors (Type I and II).
This advancement in qPCR methodology can lead to more reliable scientific findings in molecular biology research.