Molecular analysis of mouse T cell receptor expression using PCR.
This unit describes the use of the polymerase chain reaction (PCR) to characterize rearranged murine T cell receptor (TCR) transcripts present in primary lymphoid tissues, peripheral lymphoid tissues, and extra-lymphoid sites of inflammation, and to quantify them either relatively or absolutely. This methodology has been used extensively to characterize TCR variability in murine thymus and liver in T cell clones, and in extralymphoid tissues where the absolute number of T cells is limited. A procedure for cloning and sequencing PCR-amplified cDNA transcripts is provided to examine the junctional diversity of expressed genes. Also included is a method for reverse transcription of RNA into cDNA that is optimal for analysis of tissue samples where the number of T cells is limited. The detailed protocols are followed by a commentary that discusses strategies and artifacts as well as providing troubleshooting suggestions for PCR amplification and TCR analysis.
Fox
Casey J
CJ
Hospital for Sick Children Research Institute and University of Toronto, Toronto, Canada.
Danska
Jayne S
JS
Hospital for Sick Children Research Institute and University of Toronto, Toronto, Canada.
D000818:Animals; D018076:DNA, Complementary / Q000235:genetics* / Q000276:immunology; D007249:Inflammation / Q000235:genetics / Q000276:immunology / Q000473:pathology; D008099:Liver / Q000166:cytology / Q000276:immunology; D051379:Mice; D012313:RNA / Q000235:genetics / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D013950:Thymus Gland / Q000166:cytology / Q000276:immunology
D018076:DNA, Complementary; D011948:Receptors, Antigen, T-Cell; D012313:RNA