Additional TCRV beta primers and minor method modifications improve detection of clonal T-cell populations by RT-PCR.
The TCRV beta RT-PCR method for detection of clonal populations of T cells which we described previously could not detect clones that used certain variable (V) beta region families. V beta 2, 4, 8.3, and 18 had insufficient homology with the original consensus V region primer. Two new primers have been designed which work well and are able to amplify from V beta families previously undetectable by this RT-PCR. In addition, minor alterations to the cDNA synthesis and gel analysis of the PCR products make the results even easier to interpret. All the Diversity/Joining (D/J) region primer combinations except for D2/J2 have been omitted, and terminating the reverse transcription by heating prior to PCR greatly improves amplification with these primers. Use of 8% and/or 10% polyacrylamide gels increases clarity. Inclusion of the modifications described will reduce false reporting of patients as having a polyclonal T-cell population.
Lynas
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Department of Haematology, Derriford Hospital, Plymouth.
Howe
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D002999:Clone Cells / Q000276:immunology; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D016176:T-Lymphocyte Subsets / Q000276:immunology*
D018076:DNA, Complementary; D007135:Immunoglobulin Variable Region; D016693:Receptors, Antigen, T-Cell, alpha-beta