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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: Mar 29, 2026

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Published on: March 30, 2015

Understanding the qPCR Standard Curve: From Assay Validation to Absolute Quantification and Variance PCR.

Mikael Kubista^1,2,3, Amin Forootan³, Michael W Pfaffl⁴

¹Institute of Biotechnology, Czech Academy of Science, 252 42 Vestec, Czech Republic.

International Journal of Molecular Sciences

|March 28, 2026

Summary

This summary is machine-generated.

This review details quantitative PCR (qPCR) standard curve construction and analysis. A novel "variance PCR" method is proposed for estimating target molecules, potentially enhancing absolute quantification accuracy.

Keywords:

MIQE PCR RT-PCR molecular diagnostics qPCR real-time PCR standard curve varPCR variation PCR

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Related Experiment Videos

Last Updated: Mar 29, 2026

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

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Published on: March 30, 2015

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

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Published on: November 5, 2020

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

Published on: July 9, 2014

Area of Science:

Molecular Biology
Biochemistry
Analytical Chemistry

Background:

The quantitative polymerase chain reaction (qPCR) standard curve is crucial for assay validation and sample quantification.
Understanding its construction and analysis is key for accurate molecular biology experiments.

Purpose of the Study:

To provide a comprehensive explanation of qPCR standard curve generation, validation, and analysis.
To introduce and theoretically explore a novel method, "variance PCR," for absolute quantification.

Main Methods:

Examination of an idealized qPCR standard curve with high replicates to illustrate fundamental principles.
Analysis of a representative standard curve from routine workflows, including outlier assessment.
Theoretical development of "variance PCR" using replicate variation for quantification.

Main Results:

The idealized curve clarifies PCR efficiency, limit of detection, and limit of quantification.
Routine curve analysis involves dynamic range validation, outlier impact assessment, and efficiency estimation.
Variance PCR shows theoretical potential to complement digital PCR and extend quantification dynamic range.

Conclusions:

Standard curves are versatile tools in qPCR for validation and quantification.
Variance PCR offers a novel theoretical approach to enhance absolute quantification in qPCR.
This review provides an educational framework for understanding and applying qPCR standard curves.