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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: Jul 13, 2026

Isolation and Quantification of Axonal mRNAs Using Porous Membrane Inserts and RTddPCR

Isolation and Quantification of Axonal mRNAs Using Porous Membrane Inserts and RTddPCR

Published on: February 6, 2026

Assessment of normalization strategies for quantitative RT-PCR using microdissected tissue samples.

Heidi S Erickson¹, Paul S Albert, John W Gillespie

¹Pathogenetics Unit, Laboratory of Pathology and Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. HSE@nig.gov

Laboratory Investigation; a Journal of Technical Methods and Pathology

|July 24, 2007

Summary

Accurate gene expression analysis from microdissected tissues requires careful normalization. Endogenous control genes provide the most precise method for quantitative reverse transcriptase (qRT)-PCR normalization in these samples.

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Last Updated: Jul 13, 2026

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Published on: February 6, 2026

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

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Published on: June 20, 2018

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

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Published on: May 9, 2019

Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Quantitative reverse transcriptase (qRT)-PCR is a key technique for gene expression measurement.
Normalization is essential for accurate comparisons across biological samples.
Measuring gene expression in limited amounts of mRNA from microdissected (MD) tissues presents challenges.

Purpose of the Study:

To evaluate different normalization strategies for qRT-PCR in microdissected samples.
To determine the most precise method for gene expression analysis in limited tissue samples.

Main Methods:

Comparison of three normalization approaches: cell count, total RNA measurement, and endogenous control genes.
Application of these methods to microdissected tissue samples for qRT-PCR analysis.

Main Results:

Cell count and total RNA measurements are useful for initial calibration but lack precision for normalization.
Endogenous control genes accurately determine target gene abundance relative to the transcriptome.
Precise gene expression measurements are achievable with appropriate normalization.

Conclusions:

Endogenous control genes are the preferred normalization strategy for qRT-PCR in microdissected samples.
Proper normalization ensures reliable gene expression data from challenging sample types.
This study validates a robust method for gene expression profiling of MD tissues.