title: Validation of a quantitative polymerase chain reaction method for human Alu gene detection in microchimeric pigs used as donors for xenotransplantation.
abstract: This work was undertaken to evaluate whether a real-time quantitative polymerase chain reaction (qPCR) is as an adequate method for detection and quantification of human-specific DNA elements (Alu gene) in tissues and blood samples of pigs in which human stem cells were engrafted. Real-time qPCR quantification was performed with the use of previously described primers. The human DNA was mixed with different quantities of porcine DNA. The primer concentration and specificity, the qPCR efficiency, the quantification variations due to different porcine DNA concentrations, and the dissociation curve produced by the assay were evaluated. The qPCR proved to be specific, robust, with a reproducible and specific bimodal melting curve. High porcine DNA concentration produced subquantification, especially with low human DNA quantity. However, the assay proved to be useful for the detection of chimeric piglets produced by human cells injected in utero, because the effect caused by the porcine DNA interference was corrected in quantification of human DNA from piglets.
authors: Grupo de Investigación Cría y Salud Animal, Universidad de Murcia, Murcia, Spain.
authors: J M
authors: JM
authors: Abellaneda
authors: Grupo de Investigación Cría y Salud Animal, Universidad de Murcia, Murcia, Spain; Departamento de Cirugía, Hospital Clínico Universitario Virgen de la Arrixaca, Murcia, Spain.
authors: L
authors: L
authors: Martínez-Alarcón
authors: Grupo de Investigación Cría y Salud Animal, Universidad de Murcia, Murcia, Spain.
authors: J J
authors: JJ
authors: Quereda
authors: Grupo de Investigación Cría y Salud Animal, Universidad de Murcia, Murcia, Spain.
authors: J M
authors: JM
authors: Herrero-Medrano
authors: Grupo de Investigación Cría y Salud Animal, Universidad de Murcia, Murcia, Spain; Escola de Veterinaria, Universidad de Federal de Goiás, Goiás, Brazil.
authors: L
authors: L
authors: Mendonça
authors: Departamento de Inmunología. Hospital Clínico Universitario Virgen de la Arrixaca, Murcia, Spain.
authors: A
authors: A
authors: Mrowiec
authors: Departamento de Anatomía y Anatomía Patológica Comparadas, Universidad de Murcia, Murcia, Spain.
authors: O
authors: O
authors: García-Nicolás
authors: Departamento de Anatomía y Anatomía Patológica Comparadas, Universidad de Murcia, Murcia, Spain.
authors: F J
authors: FJ
authors: Pallarés
authors: Departamento de Cirugía, Hospital Clínico Universitario Virgen de la Arrixaca, Murcia, Spain.
authors: A
authors: A
authors: Ríos
authors: Grupo de Investigación Cría y Salud Animal, Universidad de Murcia, Murcia, Spain; Departamento de Producción Animal, Universidad de Murcia, Murcia, Spain.
authors: A
authors: A
authors: Muñoz
authors: Departamento de Cirugía, Hospital Clínico Universitario Virgen de la Arrixaca, Murcia, Spain.
authors: P
authors: P
authors: Ramírez
authors: Grupo de Investigación Cría y Salud Animal, Universidad de Murcia, Murcia, Spain; Departamento de Producción Animal, Universidad de Murcia, Murcia, Spain. Electronic address: guiramis@um.es.
authors: G
authors: G
authors: Ramis
mesh_terms: D020087:Alu Elements / Q000235:genetics*; D000818:Animals; D046528:Chimerism; D004247:DNA / Q000032:analysis; D017931:DNA Primers / Q000235:genetics*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine; D014183:Transplantation, Heterologous
chemical_list: D017931:DNA Primers; D004247:DNA