Uhrberg M, Wernet P
Bone Marrow Donor Center, MED-Heinrich-Heine University, Düsseldorf, Germany.
J Immunol Methods. 1996 Aug 14;194(2):155-68. doi: 10.1016/0022-1759(96)00082-8.
A novel quantitative protocol was developed for the measurement of the relative expression levels of the human TCRBV genes based on reverse transcription (RT) and subsequent competitive polymerase chain reaction (cPCR). Competitor DNA templates for the analysis of 24 different TCRBV families were generated by a simple and rapid one-step PCR procedure with a special PCR primer, which introduces a deletion in the constant region gene segment. A defined amount of TCRBV family-specific competitor DNA and the reverse transcribed cDNA of interest were amplified in the same tube with the same primer pair in a competitive way. The resulting fragments were separated on agarose gels and the densitometrical values were evaluated directly without the requirement for additional hybridization steps. For all of the 24 different TCRBV family-specific cPCRs equal amplification efficiencies were demonstrated by titration experiments for wild-type and competitor templates. For TCRBV repertoire studies, a short form of the cPCR assay was performed, requiring only one cPCR for quantitation of each TCRBV family. The exact initial amount of wild-type template in each cPCR was interpolated from TCRBV family-specific reference calibration curves. The RT-cPCR assay was applied for the quantitative assessment of the TCRBV repertoire of CD4+ and CD8+ T cell subsets in unstimulated PBMC and compared to flow cytometric analyses with a panel of monoclonal antibodies specific for TCRBV determinants. The RT-cPCR experiments revealed a differential expression of several BV families in either the CD4+ or the CD8+ fraction. The TCRBV family-specific cPCR assay presented here combines the simplicity and speed of conventional TCRBV family-specific PCR with the quantitative features of competitive PCR. TCRBV family-specific RT-cPCR has a broad application for all kinds of quantitative T cell repertoire studies and could be easily adapted for the usage with different BV-specific primer sets.
基于逆转录（RT）和随后的竞争性聚合酶链反应（cPCR），开发了一种用于测量人类TCRBV基因相对表达水平的新型定量方案。通过一种简单快速的一步PCR程序，使用特殊的PCR引物生成用于分析24个不同TCRBV家族的竞争DNA模板，该引物会在恒定区基因片段中引入缺失。将一定量的TCRBV家族特异性竞争DNA和感兴趣的逆转录cDNA在同一管中用相同的引物对进行竞争性扩增。所得片段在琼脂糖凝胶上分离，直接评估光密度值，无需额外的杂交步骤。通过对野生型和竞争模板的滴定实验证明，对于所有24种不同的TCRBV家族特异性cPCR，扩增效率均相同。对于TCRBV库研究，进行了一种简短形式的cPCR分析，每个TCRBV家族定量仅需一次cPCR。从TCRBV家族特异性参考校准曲线中内插每个cPCR中野生型模板的确切初始量。将RT-cPCR分析应用于未刺激的外周血单核细胞（PBMC）中CD4 +和CD8 + T细胞亚群的TCRBV库的定量评估，并与使用一组针对TCRBV决定簇的单克隆抗体的流式细胞术分析进行比较。RT-cPCR实验揭示了几个BV家族在CD4 +或CD8 +组分中的差异表达。本文介绍的TCRBV家族特异性cPCR分析结合了传统TCRBV家族特异性PCR的简单性和速度以及竞争性PCR的定量特征。TCRBV家族特异性RT-cPCR在各种定量T细胞库研究中具有广泛应用，并且可以轻松适用于与不同BV特异性引物组一起使用。
