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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: Jan 15, 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

A Beginner Step-by-Step Guide for Gene Expression Analysis Using QIAcuity Digital PCR.

Jonathan Weiss¹, Gerald Schock², Afif Abdel Nour²

¹QIAGEN GmbH, Hilden, Germany. Jonathan.Weiss@qiagen.com.

Methods in Molecular Biology (Clifton, N.J.)

|October 10, 2025

Summary

This summary is machine-generated.

Digital PCR (dPCR) provides precise gene expression analysis by partitioning reactions for absolute quantification. This method overcomes quantitative PCR (qPCR) limitations, offering improved accuracy and inhibitor resistance for novel research avenues.

Keywords:

Digital PCR (dPCR)Gene expression (Gex)Multiplexing QIAcuity RNA Quantification

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Last Updated: Jan 15, 2026

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Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Gene expression (GEX) is a fundamental biological process involving messenger ribonucleic acid (mRNA) crucial for cellular function.
Tight regulation of GEX is vital for development and environmental responses; aberrant GEX is linked to diseases.
Quantitative PCR (qPCR) is a common method, but has limitations regarding efficiency, inhibitors, and low-abundance targets.

Purpose of the Study:

To highlight the advantages of Digital PCR (dPCR) for gene expression analysis.
To compare dPCR with quantitative PCR (qPCR) in terms of precision, accuracy, and limitations.
To demonstrate how dPCR enables previously inaccessible research in gene expression.

Main Methods:

Digital PCR (dPCR) utilizes reaction partitioning for absolute nucleic acid quantification without standard curves.
Comparison of dPCR and qPCR performance, focusing on accuracy, variability, and inhibitor resistance.
Analysis of low-abundance target quantification and performance across different expression levels.

Main Results:

dPCR offers highly precise absolute quantification of nucleic acids, overcoming qPCR's need for standard curves.
dPCR minimizes variability and artifacts associated with low-abundance targets, unlike qPCR.
dPCR demonstrates greater accuracy and resistance to inhibitors compared to qPCR.

Conclusions:

dPCR provides superior accuracy and robustness for gene expression analysis, especially for low-abundance targets.
dPCR overcomes key limitations of qPCR, enabling more reliable and reproducible results.
The capabilities of dPCR empower researchers to investigate gene expression in ways not feasible with qPCR.