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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: Jun 25, 2026

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

Published on: July 29, 2019

Evaluating precision and accuracy when quantifying different endogenous control reference genes in maize using

Tandace A Scholdberg¹, Tim D Norden, Daishia D Nelson

¹Grain Inspection, Packers and Stockyards Administration, Technical Services Division, U.S. Department of Agriculture, 10383 North Ambassador Drive, Kansas City, Missouri 64153, USA.

Journal of Agricultural and Food Chemistry

|March 12, 2009

Summary

Evaluating endogenous control genes for real-time quantitative PCR (RT-qPCR) in maize revealed variable performance across cultivars. Identifying a consistent control gene is crucial for harmonizing the detection of biotechnology-derived traits in maize.

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Last Updated: Jun 25, 2026

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

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Area of Science:

Agricultural biotechnology
Molecular biology
Plant genetics

Background:

Real-time quantitative PCR (RT-qPCR) is essential for detecting biotechnology-derived traits in plants, meeting regulatory requirements for DNA trace detection.
Accurate quantification relies on normalizing target gene copy number with an endogenous control gene, as per ISO 21570:2005 standards.

Purpose of the Study:

To assess the predictability and performance of commonly used endogenous control genes for detecting biotechnology-derived traits in maize.
To identify a suitable endogenous control gene that amplifies consistently across diverse maize cultivars.

Main Methods:

Real-time qPCR was utilized to evaluate the amplification efficiency of several endogenous control genes (starch synthase, alcohol dehydrogenase, high-mobility group, zein, and invertase).
Performance was assessed by comparing isogenic maize to certified reference standards and nonisogenic maize cultivars to a reference standard (IRMM Bt-11).

Main Results:

Accurate and precise amplification efficiencies were observed when comparing isogenic maize to certified reference standards.
Highly variable results were obtained when 23 nonisogenic maize cultivars were compared to the IRMM Bt-11 reference standard, indicating cultivar-dependent performance.

Conclusions:

The choice of endogenous control gene significantly impacts the reliability of RT-qPCR for detecting biotechnology-derived traits in maize.
Establishing an internationally recognized, consistently amplifying endogenous control gene is vital for harmonizing testing protocols and ensuring accurate trait detection across different maize varieties.