Zhou Dun, Srivastava Rajneesh, Grummel Verena, Cepok Sabine, Hartung Hans-Peter, Hemmer Bernhard
Neuroimmunology Group, Department of Neurology, Heinrich Heine-University, Duesseldorf, Germany.
Lab Invest. 2006 Mar;86(3):314-21. doi: 10.1038/labinvest.3700381.
Analysis of T-cell receptor beta chain (TCR-beta) rearrangement is essential to investigate T-cell responses in human autoimmune diseases, infection and cancer. Since the TCR-beta locus contains 55 variable (V) region gene segments, multiple assays have been necessary to determine TCR-beta rearrangements of individual T cells. We established a seminested rtPCR method for single T-cell analysis with two sets of degenerate primers covering 76 and 24% of the TCR-Vbeta genes, respectively. The specificity of the approach was validated by screening cDNAs obtained from T-cell clones (TCC) with defined TCR-beta rearrangement. We applied the method successfully to profile TCR-beta rearrangement of single T cells sorted from body fluids or dissected tissue. Concomitant analysis of other gene transcripts allowed determining phenotype and function of TCR-beta-defined single T cells. Our fast, cost-efficient and high throughput approach will facilitate studies on T-cell responses in human diseases.
分析T细胞受体β链（TCR-β）重排对于研究人类自身免疫性疾病、感染和癌症中的T细胞反应至关重要。由于TCR-β基因座包含55个可变（V）区基因片段，因此需要多种检测方法来确定单个T细胞的TCR-β重排。我们建立了一种半巢式rtPCR方法用于单个T细胞分析，使用两组简并引物，分别覆盖76%和24%的TCR-Vβ基因。通过筛选从具有明确TCR-β重排的T细胞克隆（TCC）获得的cDNA，验证了该方法的特异性。我们成功地将该方法应用于分析从体液或解剖组织中分选的单个T细胞的TCR-β重排。对其他基因转录本的同步分析有助于确定TCR-β定义的单个T细胞的表型和功能。我们快速、经济高效且高通量的方法将促进对人类疾病中T细胞反应的研究。
