Accurate and objective copy number profiling using real-time quantitative PCR

Abstract
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 presentation, all relevant information for a successful implementation of qPCR based copy number analysis will be given. 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 are emphasized. Furthermore, we suggest an appropriate and practical way to calculate copy numbers and to objectively interpret the results. The guidelines provided in this presentation will most certainly improve the quality and reliability of your qPCR based copy number screening, and allow it to be implemented in a diagnostic setting or a high-throughput screening.