Sample degradation leads to false-positive copy number variation calls in multiplex real-time polymerase chain reaction assays

Miami Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136, USA.
Analytical Biochemistry (Impact Factor: 2.31). 03/2009; 386(2):288-90. DOI: 10.1016/j.ab.2008.11.040
Source: PubMed

ABSTRACT The recent implication of genomic copy number variations (CNVs) in multiple human genetic disorders has led to increased interest in CNV discovery technologies. There is a growing consensus that, in addition to the method used for detection, at least one additional technology should be employed for validation. Real-time quantitative polymerase chain reaction (qPCR) analysis, incorporating a normal (2N) copy number standard, is commonly used as a means of validating CNVs. Whereas it has previously been reported that formalin-fixed paraffin-embedded (FFPE) DNA samples can yield spurious CNV calls in real-time qPCR assays, here we report that sample degradation under standard laboratory storage conditions generates a significant increase in false-positive CNV results. Results suggest the possibility of biased degradation among genomic regions and emphasize the need to assess sample integrity immediately prior to real-time qPCR experiments.

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    • "This may also be reflected in expression studies, where target and reference differ in several magnitudes. Dependency on short amplicons for reliable measurements on degraded RNA [20] and false-positive copy number calls in multiplexed qPCR assays in degraded samples [33] have been reported. "
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    • "Combining results obtained with DNA isolated from other sources or using other extraction protocols was not attempted because SQM-PCR results could be affected by different DNA qualities [data not shown and Charbonnier et al., 2000; Heath et al., 2000; Cukier et al., 2009]. Briefly, 2 internal control regions known to have no CNV and regions of interest were co-amplified in multiplex PCR under quantitative PCR conditions optimising PCR protocols and primer amount. "
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