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Published on: April 25, 2011
Real-time reverse-transcription polymerase chain reaction: technical considerations for gene expression analysis
Shareen H Doak1, Zoulikha M Zaïr
1DNA Damage Research Group, Institute of Life Science, College of Medicine, Swansea University, Swansea, Wales, UK. s.h.doak@swansea.ac.uk
Methods in Molecular Biology (Clifton, N.J.)
|December 8, 2011
Summary
Reverse transcription-polymerase chain reaction (RT-PCR) quantifies mRNA levels using retroviral enzymes and PCR. This sensitive method allows for relative or absolute gene expression analysis, even with limited RNA samples.
Area of Science:
- Molecular Biology
- Gene Expression Analysis
Background:
- Reverse transcription-polymerase chain reaction (RT-PCR) is a sensitive technique for quantifying mRNA.
- It is particularly useful for samples with limited RNA or low-level transcripts.
Purpose of the Study:
- To describe popular RT-PCR techniques.
- To detail methodologies for analyzing RT-PCR data for gene expression quantification.
Main Methods:
- Utilizes retroviral reverse transcriptase (RT) activity coupled with PCR.
- Amplifies specific mRNA transcripts.
- Incorporates fluorescence detection for amplicon quantification.
Main Results:
- PCR product concentration is directly proportional to initial mRNA quantity.
- Enables quantification of gene expression levels.
Conclusions:
- RT-PCR is a versatile tool for gene expression studies.
- Provides methods for both relative and absolute quantification of mRNA levels.
