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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...

PCR

PCR

PCR - Polymerase Chain Reaction

PCR - Polymerase Chain Reaction

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Related Experiment Video

Updated: Jun 18, 2026

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Published on: February 25, 2017

How to do successful gene expression analysis using real-time PCR.

Stefaan Derveaux¹, Jo Vandesompele, Jan Hellemans

¹Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Methods (San Diego, Calif.)

|December 9, 2009

Summary

This summary is machine-generated.

Ensuring quality control throughout the reverse transcription quantitative PCR (RT-qPCR) workflow is crucial for accurate gene expression analysis. Addressing critical issues from planning to reporting guarantees biologically meaningful results.

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Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Reverse transcription quantitative PCR (RT-qPCR) is a widely used method for gene expression analysis.
Despite its accuracy and sensitivity, numerous critical issues can compromise RT-qPCR results.
Ensuring reliable data requires careful attention to the entire experimental workflow.

Purpose of the Study:

To review the complete RT-qPCR workflow, from planning to reporting.
To highlight critical steps and potential pitfalls in RT-qPCR experiments.
To emphasize the importance of quality assurance and control in RT-qPCR.

Main Methods:

Comprehensive literature review of RT-qPCR best practices.
Analysis of the RT-qPCR workflow, broken down into Plan/Prepare, Cycle, and Report (PCR) phases.
Identification of key quality control checkpoints throughout the process.

Main Results:

Critical issues exist at every stage of the RT-qPCR workflow, impacting data reliability.
Quality assurance and control are essential from initial planning and sample preparation to data analysis and reporting.
A systematic approach (PCR: plan/prepare, cycle, report) is recommended for robust RT-qPCR.

Conclusions:

Biologically meaningful and trustworthy conclusions from RT-qPCR depend on rigorous quality control.
Addressing potential errors in nucleic acid extraction, enzymatic steps, and data analysis is vital.
Implementing comprehensive quality assurance throughout the RT-qPCR workflow is paramount for accurate gene expression measurement.