title: A semi-nested real-time PCR method to detect low chimerism percentage in small quantity of hematopoietic stem cell transplant DNA samples.
abstract: Chimerism status evaluation of post-allogeneic hematopoietic stem cell transplantation samples is essential to predict post-transplant relapse. The most commonly used technique capable of detecting small increments of chimerism is quantitative real-time PCR. Although this method is already used in several laboratories, previously described protocols often lack sensitivity and the amount of the DNA required for each chimerism analysis is too high. In the present study, we compared a novel semi-nested allele-specific real-time PCR (sNAS-qPCR) protocol with our in-house standard allele-specific real-time PCR (gAS-qPCR) protocol. We selected two genetic markers and analyzed technical parameters (slope, y-intercept, R2, and standard deviation) useful to determine the performances of the two protocols. The sNAS-qPCR protocol showed better sensitivity and precision. Moreover, the sNAS-qPCR protocol requires, as input, only 10 ng of DNA, which is at least 10-fold less than the gAS-qPCR protocols described in the literature. Finally, the proposed sNAS-qPCR protocol could prove very useful for performing chimerism analysis with a small amount of DNA, as in the case of blood cell subsets.
authors.affiliation: a Department of Life Sciences, University of Trieste, Trieste, Italy; Medical Genetics, Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste, Italy.
authors.forename: Michelangelo
authors.initials: M
authors.lastname: Aloisio
authors.affiliation: b Medical Genetics, Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Bortot
authors.affiliation: c Department of Medical Sciences, University of Trieste, Trieste, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Gandin
authors.affiliation: b Medical Genetics, Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste, Italy.
authors.forename: Giovanni Maria
authors.initials: GM
authors.lastname: Severini
authors.affiliation: b Medical Genetics, Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste, Italy.
authors.forename: Emmanouil
authors.initials: E
authors.lastname: Athanasakis
mesh_terms: D000483:Alleles; D005819:Genetic Markers; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D011110:Polymorphism, Genetic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D018183:Transplantation Chimera / Q000235:genetics*; D014184:Transplantation, Homologous
chemical_list: D005819:Genetic Markers
keywords: HSCT allogénique; PCR en temps réel semi-nichée; Sybr Green; allogeneic HSCT; chimerism; chimérisme; semi-nested real-time PCR