Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.
Methods. 2010 Apr;50(4):262-70. doi: 10.1016/j.ymeth.2009.12.007. Epub 2010 Jan 6.
Copy number changes are known to be involved in numerous human genetic disorders. In this context, qPCR-based copy number screening may serve as the method of choice for targeted screening of the relevant disease genes and their surrounding regulatory landscapes. qPCR has many advantages over alternative methods, such as its low consumable and instrumentation costs, fast turnaround and assay development time, high sensitivity and open format (independent of a single supplier). In this chapter we provide all relevant information for a successfully implement of qPCR-based copy number analysis. We emphasize the significance of thorough in silico and empirical validation of the primers, the need for a well thought-out experiment design, and the importance of quality controls along the entire workflow. Furthermore, we suggest an appropriate and practical way to calculate copy numbers and to objectively interpret the results. The provided guidelines will most certainly improve the quality and reliability of your qPCR-based copy number screening.
拷贝数变化已知涉及许多人类遗传疾病。在这种情况下，基于 qPCR 的拷贝数筛选可能是针对相关疾病基因及其周围调控景观进行靶向筛选的首选方法。qPCR 相对于其他方法具有许多优势，例如耗材和仪器成本低、周转时间和检测开发时间快、灵敏度高以及格式开放（不依赖于单一供应商）。在本章中，我们提供了成功实施基于 qPCR 的拷贝数分析所需的所有相关信息。我们强调了彻底的计算机模拟和引物验证的重要性、精心设计实验的必要性以及整个工作流程中质量控制的重要性。此外，我们还提出了一种计算拷贝数并客观解释结果的合适且实用的方法。提供的指南肯定会提高基于 qPCR 的拷贝数筛选的质量和可靠性。
