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...

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Bibliographic Details
Published in:Transplantation proceedings 2015, Vol.47 (1), p.132-135
Main Authors: Abellaneda, J.M, Martínez-Alarcón, L, Quereda, J.J, Herrero-Medrano, J.M, Mendonça, L, Mrowiec, A, García-Nicolás, O, Pallarés, F.J, Ríos, A, Muñoz, A, Ramírez, P, Ramis, G
Format: Article
Language:English
Subjects:
Alu Elements - genetics
Animals
Chimerism
DNA - analysis
DNA Primers - genetics
Humans
Real-Time Polymerase Chain Reaction - methods
Sensitivity and Specificity
Surgery
Swine
Transplantation, Heterologous
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Summary: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.
ISSN:0041-1345
1873-2623
DOI:10.1016/j.transproceed.2014.11.016