Walters Giles, Alexander Stephen I
Department of Nephrology, Leicester General Hospital, Gwendolen Road, Leicester LE5 4PW, United Kingdom.
J Immunol Methods. 2004 Nov;294(1-2):43-52. doi: 10.1016/j.jim.2004.08.015. Epub 2004 Oct 6.
Real time PCR is a useful tool in immunological research but little has been published on the use of this technique in the measurement of T cell receptor (TCR) BV repertoires. We have compared the performance of SYBR green with that of a dual-labeled HuTrec (Human T cell receptor) fluorescent probe system. Serial dilutions of peripherals blood mononuclear cells were tested to compare the consistency of the two systems across multiple T cell receptor signal levels. Samples were diluted with non-TCR cDNA to simulate a low-level TCR signal within a tissue sample. The fluorogenic probe gave highly consistent results with a correlation coefficient of greater than 0.9 across a samples. SYBR green showed accurate results only when tested with high signal samples. Low-signal cDNA gave very poor results with SYBR green compared to the HuTrec fluorogenic probe with correlation coefficients as low as 0.65. Poorest performance occurred in the context of the simulated tissue sample with a high level of non-TCR DNA. Under these conditions, large amounts of nonspecific PCR product were generated which were detected by the SYBR green system and therefore distorted the results. SYBR green performed poorly when used with samples contaminated with significant quantities of non-target cDNA and samples with low target signal. It is therefore not an appropriate method for the measurement of TCR repertoires in small tissue samples. A dual-labeled HuTrec fluorescent probe produced a consistent TCR repertoire across a broad range of TCR signal levels and proved robust in the presence of contaminating non-TCR cDNA. We recommend the use of such a fluorescent probe in real time PCR for the assessment of TCR repertoires in small tissue samples. Where samples are assayed using SYBR green, agarose gel confirmation of PCR product specificity should be provided.
实时聚合酶链反应(Real time PCR)是免疫研究中的一种有用工具,但关于该技术在测量T细胞受体(TCR)BV基因库方面的应用,发表的文献较少。我们比较了SYBR Green与双标记HuTrec(人类T细胞受体)荧光探针系统的性能。对外周血单个核细胞进行系列稀释,以比较这两种系统在多个T细胞受体信号水平上的一致性。用非TCR cDNA稀释样品,以模拟组织样品中的低水平TCR信号。荧光探针给出了高度一致的结果,样品间的相关系数大于0.9。SYBR Green仅在检测高信号样品时显示出准确的结果。与HuTrec荧光探针相比,低信号cDNA用SYBR Green检测时结果很差,相关系数低至0.65。在含有高水平非TCR DNA的模拟组织样品中,性能最差。在这些条件下,产生了大量非特异性PCR产物,SYBR Green系统检测到这些产物,从而扭曲了结果。当SYBR Green与大量非靶标cDNA污染的样品以及低靶标信号的样品一起使用时,表现不佳。因此,它不是测量小组织样品中TCR基因库的合适方法。双标记HuTrec荧光探针在广泛的TCR信号水平上产生了一致的TCR基因库,并且在存在污染性非TCR cDNA的情况下表现出稳健性。我们建议在实时PCR中使用这种荧光探针来评估小组织样品中的TCR基因库。如果使用SYBR Green对样品进行检测,应提供PCR产物特异性的琼脂糖凝胶确认。