Translational Medicine Research group, Cranfield Health, Cranfield University, Cranfield, UK.
J Immunol Methods. 2013 Mar 29;389(1-2):1-8. doi: 10.1016/j.jim.2012.12.003. Epub 2013 Jan 9.
The level of T-cell receptor excision circles (TREC), which decline with advancing age in normal individuals, has recently gained interest as a reference marker for studies on premature or early immunosenescence under particular health conditions. In order to facilitate translational studies at population and clinical levels, essential for the understanding of how changes in TREC levels are associated with responsiveness of the immune system, we have developed and optimized a real-time polymerase chain reaction (qPCR) assay which quantifies the TREC ratio from dried blood spot (DBS) samples. The present study considers a fully automated procedure to purify DNA and amplify sequences of interests by means of qPCR from DBS samples collected in healthy adults. Both TREC:PBMC (peripheral blood mononuclear cell) and TREC:T-cell ratios were compared for intra- and inter individual reproducibility. Furthermore, the impact of the length of storage on the quality of the DNA generated was also analyzed. In conclusion we describe a fully automated procedure for extracting DNA and qPCR set up, which offers a high-precision, robust qPCR assay for the quantification of both TREC:T-cell ratio and TREC:PBMC from DBS samples.
T 细胞受体切除环（TREC）的水平随着正常个体年龄的增长而下降，最近作为特定健康状况下研究过早或早期免疫衰老的参考标志物引起了关注。为了促进人群和临床水平的转化研究，这对于理解 TREC 水平的变化与免疫系统的反应性之间的关系至关重要，我们开发并优化了一种实时聚合酶链反应（qPCR）检测方法，可从干血斑（DBS）样本中定量 TREC 比值。本研究考虑了一种全自动程序，通过 qPCR 从健康成年人采集的 DBS 样本中纯化 DNA 并扩增感兴趣的序列。比较了 TREC：PBMC（外周血单核细胞）和 TREC：T 细胞比值的个体内和个体间重现性。此外，还分析了储存时间长短对生成 DNA 质量的影响。总之，我们描述了一种全自动提取 DNA 和 qPCR 建立的方法，该方法提供了一种高精度、稳健的 qPCR 检测方法，可从 DBS 样本中定量 TREC：T 细胞比值和 TREC：PBMC。
