title: Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real-time PCR.
abstract: Taking into account the importance of goats as transgenic models, as well as the rarity of copy number (CN) studies in farm animals, the present work aimed to evaluate methodological strategies for accurate and precise transgene CN quantification in goats using quantitative polymerase chain reaction (qPCR). Mouse and goat lines transgenic for human granulocyte-colony stimulating factor were used. After selecting the best genomic DNA extraction method to be applied in mouse and goat samples, intra-assay variations, accuracy and precision of CN quantifications were assessed. The optimized conditions were submitted to mathematical strategies and used to quantify CN in goat lines. The findings were as follows: validation of qPCR conditions is required, and amplification efficiency is the most important. Absolute and relative quantifications are able to produce similar results. For normalized absolute quantification, the same plasmid fragment used to generate goat lines must be mixed with wild-type goat genomic DNA, allowing the choice of an endogenous reference gene for data normalization. For relative quantifications, a resin-based genomic DNA extraction method is strongly recommended when using mouse tail tips as calibrators to avoid tissue-specific inhibitors. Efficient qPCR amplifications (≥95%) allow reliable CN measurements with SYBR technology. TaqMan must be used with caution in goats if the nucleotide sequence of the endogenous reference gene is not yet well understood. Adhering to these general guidelines can result in more exact CN determination in goats. Even when working under nonoptimal circumstances, if assays are performed that respect the minimum qPCR requirements, good estimations of transgene CN can be achieved.
authors.affiliation: Laboratory of Physiology and Control of Reproduction, Faculty of Veterinary, State University of Ceará, Fortaleza, Brazil.
authors.forename: Ribrio I T P
authors.initials: RI
authors.lastname: Batista
authors.forename: Maria C S
authors.initials: MC
authors.lastname: Luciano
authors.forename: Dárcio I A
authors.initials: DI
authors.lastname: Teixeira
authors.forename: Vicente J F
authors.initials: VJ
authors.lastname: Freitas
authors.forename: Luciana M
authors.initials: LM
authors.lastname: Melo
authors.forename: Lyudmila E
authors.initials: LE
authors.lastname: Andreeva
authors.forename: Irina A
authors.initials: IA
authors.lastname: Serova
authors.forename: Oleg L
authors.initials: OL
authors.lastname: Serov
mesh_terms: D000818:Animals; D004247:DNA / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D005456:Fluorescent Dyes; D018628:Gene Dosage / Q000235:genetics*; D006041:Goats / Q000235:genetics*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
chemical_list: D005456:Fluorescent Dyes; D004247:DNA
keywords: SYBR Green; TaqMan; copy number; real-time PCR; transgenic mice