Journal
METHODS
Volume 50, Issue 4, Pages 262-270Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2009.12.007
Keywords
qPCR; CNV; Copy number variations; Validation; Experiment design; Quality control
Funding
- Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen)
- Research Foundation Flanders (FWO) [1.2.843.07.N.1]
- BOF-UGent [01209407]
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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 silica and empirical validation of the primers, the need for a well thought-out experiment design, and the importance of quality controls along the entire work-flow. 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. (C) 2010 Elsevier Inc. All rights reserved.
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