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Molecular analysis of mouse T cell receptor expression using PCR
1Hospital for Sick Children Research Institute and University of Toronto, Toronto, Canada.
Current Protocols in Immunology
|April 25, 2008
Summary
This study details Polymerase Chain Reaction (PCR) methods for characterizing and quantifying T cell receptor (TCR) transcripts in various mouse tissues. It provides protocols for analyzing TCR variability and junctional diversity, even in samples with limited T cells.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- T cell receptor (TCR) diversity is crucial for adaptive immunity.
- Characterizing TCR transcripts in various tissues, especially those with limited T cells, presents technical challenges.
Purpose of the Study:
- To describe Polymerase Chain Reaction (PCR) methods for characterizing and quantifying rearranged murine T cell receptor (TCR) transcripts.
- To provide protocols for analyzing TCR variability and junctional diversity in diverse tissue types.
Main Methods:
- Utilizing Polymerase Chain Reaction (PCR) for amplification of T cell receptor (TCR) transcripts.
- Employing reverse transcription of RNA to cDNA for optimal analysis of limited cell samples.
- Implementing cloning and sequencing of PCR-amplified cDNA to assess junctional diversity.
Main Results:
- Established protocols for relative and absolute quantification of TCR transcripts.
- Demonstrated applicability in primary, peripheral, and extra-lymphoid tissues.
- Provided methods suitable for samples with limited T cell numbers.
Conclusions:
- The described PCR-based methodology enables comprehensive characterization of TCR transcripts.
- This approach is valuable for studying TCR variability and gene expression in various immunological contexts.
- Detailed protocols and troubleshooting guidance facilitate successful TCR analysis.
