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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: May 23, 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

Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns.

Veronica Sanchez-Freire¹, Antje D Ebert, Tomer Kalisky

¹Department of Medicine, Stanford University School of Medicine, California, USA.

Nature Protocols

|April 7, 2012

Summary

High-content microfluidic real-time quantitative real-time PCR (qRT-PCR) enables rapid gene expression analysis in single cells. This technology aids in understanding developmental processes and human pluripotent stem cell regulation.

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High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Published on: August 3, 2011

Area of Science:

Molecular Biology
Stem Cell Biology
Microfluidics

Background:

Gene expression analysis in single cells is crucial for understanding cellular heterogeneity.
Traditional methods face limitations with small sample sizes and mixed cell populations.
Investigating developmental processes requires precise molecular-level insights.

Purpose of the Study:

To present a high-content microfluidic real-time platform for single-cell gene expression analysis.
To enable comparative investigation of developmental process regulation in single cells.
To provide a standardized assay for analyzing human pluripotent stem cell self-renewal and differentiation.

Main Methods:

Utilizing single-cell quantitative real-time PCR (qRT-PCR).
Employing high-throughput arrays and microfluidic technology.
Analyzing gene expression profiles approximately 11 hours post-sample collection.

Main Results:

The platform overcomes limitations of heterogeneous cell populations and sample amounts.
Enables comparative analysis of gene expression in single cells.
Provides insights into differential gene regulation in normal versus disease contexts.

Conclusions:

High-throughput single-cell qRT-PCR is a powerful tool for developmental biology research.
This approach facilitates in-depth analysis of human pluripotent stem cell mechanisms.
The microfluidic platform offers a standardized method for complex cellular studies.