Relative quantification of TCR Vbeta -chain families by real time PCR for identification of clonal T-cell populations
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BioMed Central
Open Access
Methodology
Relative quantification of TCR Vbeta-chain families by real time PCR for identification of clonal T-cell populations Sebastian Ochsenreither*1, Alberto Fusi1, Antonia Busse1, Dirk Nagorsen1, David Schrama2, Jürgen Becker2, Eckhard Thiel1 and Ulrich Keilholz1 Address: 1University Hospital Benjamin Franklin, Medizinische Klinik III, Hematology, Oncology, and Transfusion Medicine, Charité Universitätsmedizin Berlin, 12200, Berlin, Germany and 2University of Würzburg, Clinic for Dermatology, Allergology, and Venerology, 97080, Würzburg, Germany Email: Sebastian Ochsenreither* - [email protected]; Alberto Fusi - [email protected]; Antonia Busse - [email protected]; Dirk Nagorsen - [email protected]; David Schrama - [email protected]; Jürgen Becker - [email protected]; Eckhard Thiel - [email protected]; Ulrich Keilholz - [email protected] * Corresponding author
Published: 1 July 2008 Journal of Translational Medicine 2008, 6:34
doi:10.1186/1479-5876-6-34
Received: 18 February 2008 Accepted: 1 July 2008
This article is available from: http://www.translational-medicine.com/content/6/1/34 © 2008 Ochsenreither et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract 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. Method: Vβ-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 α/β TCR was assessed by quantification of the constant α-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. Conclusion: We introduce a versatile method for the relative quantification of Vβ-families by real time PCR. The experimental strategy described allows the identification of alterations in the Vβfamily repertoire.
Background T-lymph
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