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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 22, 2026

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Published on: April 30, 2010

Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples.

Heidi S Erickson¹, Paul S Albert, John W Gillespie

¹Pathogenetics Unit, Laboratory of Pathology and Urologic Oncology Branch, National Cancer Institute, NIH, Bethesda, MD, USA.

Nature Protocols

|May 30, 2009

Summary

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) provides gene expression data from microdissected cells. This protocol optimizes qRT-PCR for small, variable samples, ensuring reliable gene expression measurements.

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Published on: April 30, 2010

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Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is essential for gene expression analysis.
Microdissected cells from animal or clinical tissues present challenges like variable RNA quality and quantity.
Standard qRT-PCR methods need adaptation for small, dissected cell populations.

Purpose of the Study:

To present an adapted protocol for reliable gene expression measurement from microdissected cells.
To address challenges in RNA quality, quantity, and normalization in small sample sets.
To enable meaningful comparisons across diverse sample sets using qRT-PCR.

Main Methods:

Tissue microdissection to isolate specific cell populations.
RNA extraction and quality/quantity assessment from limited samples.
Adaptation of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) protocols.
Development of a robust normalization strategy and data analysis pipeline.

Main Results:

A comprehensive protocol for qRT-PCR on microdissected samples is detailed.
The protocol includes specific steps for RNA handling and normalization.
Reliable gene expression measurements are achievable from small, variable cell populations.

Conclusions:

The described protocol enables accurate gene expression analysis using qRT-PCR on microdissected cells.
This method overcomes common challenges associated with limited and variable RNA samples.
The protocol facilitates reliable comparisons of gene expression across different sample sets.