accuracy in copy number calling by qpcr and prt a matter of dna拷贝数的准确性要求qpcr和prt dna.pdfVIP

Accuracy in Copy Number Calling by qPCR and PRT: A Matter of DNA 1,2 1,2 1,2 1 Nora Fernandez-Jimenez , Ainara Castellanos-Rubio , Leticia Plaza-Izurieta , Galder Gutierrez , ˜ 1,3 ˜ 1,3 1,3 1,2 Inaki Irastorza , Luis Castano , Juan Carlos Vitoria , Jose Ramon Bilbao * 1 Immunogenetics Research Laboratory, Cruces University Hospital, Barakaldo, Basque Country, Spain, 2 Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, Leioa, Basque Country, Spain, 3 Department of Pediatrics, University of the Basque Country, Bilbao, Basque Country, Spain Abstract The possible implication of copy number variation (CNV) in the genetic susceptibility to human disease needs to be assessed using robust methods that can be applied at a population scale. In this report, we analyze the performance of the two major techniques, quantitative PCR (qPCR) and paralog ratio test (PRT), and investigate the influence of input DNA amount and template integrity on the reliability of both methods. Analysis of three genes (PRELID1, SYNPO and DEFB4) in a large sample set showed that both methods are prone to false copy number assignments if sufficient attention is not paid to DNA concentration and quality. Accurate normalization of samples is essential for reproducible qPCR because it avoids the effect of differential amplification efficiencies between target and control assays, whereas PRT is generally more sensitive to template degradation due to the fact that longer ampl