Reliable and cost-effective approach for diagnosis of heterozygous F8/F9 large deletions by quantitative real-time PCR

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Bibliographic Details
Published in:	Haemophilia : the official journal of the World Federation of Hemophilia Vol. 21; no. 3; pp. e247 - e251
Main Authors:	Abelleyro, M. M., Radic, C. P., Tetzlaff, T., Marchione, V., Fundia, A. F., Larripa, I. B., Rossetti, L. C., De Brasi, C. D.
Format:	Journal Article
Language:	English
Published:	England Blackwell Publishing Ltd 01-05-2015 Wiley Subscription Services, Inc
Subjects:	Factor IX - genetics Factor VIII - genetics Hemophilia A - diagnosis Hemophilia A - genetics Hemophilia B - diagnosis Hemophilia B - genetics Humans Real-Time Polymerase Chain Reaction - economics Real-Time Polymerase Chain Reaction - methods Reproducibility of Results Sequence Deletion
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Bibliography:	René Barón Foundation Table ST1. Oligonucleotide primers for qPCR, long-range PCR and standard-PCR.Fig. SF1. Crossing threshold (Ct) vs. concentration calibration curves and Melting curves. Calibration curves made with serial dilutions of a genomic DNA sample of a male from the general healthy population (C85) by triplicate (blue dots) on the left panel and their associated melting curves on the right panel. Calibration curve parameters are shown: slope (M), amplification efficiency and R2. Row A shows amplification of F8 exon 6; B with F8 exon 26; C with F9 exon 8; D with F8 promoter; E with CTLA4a (a reference for F8 exon 6, F8 exon 26 and F9 exon 8 targets); and F with CTLA4b (a reference for F8 promoter).Fig. SF2. Specificity analysis of qPCR products by agarose gel electrophoresis. Lane 1: 100 bp marker. Lanes 2-15: qPCR products are organized as Normal Control (lanes 2, 5, 8, 11 and 14, control C85) used to performed the calibration curves, a female at risk of being carrier (lane 3, case #408; lane 6, case #137; lane 9, case #563; lane 12, case # 382) and negative controls (no DNA) (lanes 4, 7, 10, 13 and 15). qPCR products: F8 promoter (lanes 2-4); F8 exon 6 (lanes 5-7); F8 exon 26 (lanes 8-10); F9 exon 8 (lanes 11-13); and CTLA4 (lanes 14-15). ark:/67375/WNG-RQ1BVX5T-9 National Academy of Medicine of Buenos Aires Alberto J. Roemmers Foundation ArticleID:HAE12656 istex:9DD74E30C9DC13E87B2B2353F756B2D0FC25A3C0 Novo Nordisk Argentina World Federation of Hemophilia Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) National Research Council (CONICET)
ISSN:	1351-8216 1365-2516
DOI:	10.1111/hae.12656