Relative quantification of TCR Vbeta-chain families by real time PCR for identification of clonal T-cell populations.
BACKGROUND
Quantification of T-cell receptor (TCR) chain families can be utilized for detection of clonal T-cell populations. Besides southern blotting and antibody-based approaches, quantitative real time PCR (qRT PCR) has been more widely applied in this context during the last years. Here, the heterogeneity of sequences within single families is the most challenging problem for exact quantification.


METHODS
Vbeta-families were quantified using a universal reverse primer and family-specific forward primers with TaqMan technology on a light cycler instrument. Relative concentrations were calculated considering slopes and crossing points of each PCR reaction. Total expression of alpha/beta TCR was assessed by quantification of the constant alpha-chain as a further control.


RESULTS
The method was tested by serial dilutions of clonal T-cells in mononuclear cells from healthy volunteers. Calculated percentages were in good correspondence with qRT PCR results demonstrating high reliability. Duplicates showed excellent technical reproducibility. We analyzed blood samples of 20 healthy volunteers for determination of mean and standard deviation for each family. The method was applied both to tissue and blood samples from patients with carcinomas and hematological disorders.


CONCLUSIONS
We introduce a versatile method for the relative quantification of Vbeta-families by real time PCR. The experimental strategy described allows the identification of alterations in the Vbeta-family repertoire.
Ochsenreither
Sebastian
S
University Hospital Benjamin Franklin, Medizinische Klinik III, Hematology, Oncology, and Transfusion Medicine, Charité Universitätsmedizin Berlin, 12200, Berlin, Germany. sebastian.ochsenreither@charite.de
Fusi
Alberto
A
Busse
Antonia
A
Nagorsen
Dirk
D
Schrama
David
D
Becker
Jürgen
J
Thiel
Eckhard
E
Keilholz
Ulrich
U
D001483:Base Sequence; D002469:Cell Separation / Q000379:methods; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D019169:Jurkat Cells; D005810:Multigene Family; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D016693:Receptors, Antigen, T-Cell, alpha-beta