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.
| Original language | English |
|---|---|
| Pages (from-to) | 183-192 |
| Number of pages | 10 |
| Journal | Genome |
| Volume | 60 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 2017 |
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Keywords
- Allogeneic HSCT
- Chimerism
- Semi-nested real-time PCR
- Sybr Green
ASJC Scopus subject areas
- Biotechnology
- Molecular Biology
- Genetics
Cite this
A semi-nested real-time PCR method to detect low chimerism percentage in small quantity of hematopoietic stem cell transplant DNA samples. / Aloisio, Michelangelo; Bortot, Barbara; Gandin, Ilaria; Severini, Giovanni Maria; Athanasakis, Emmanouil; Xu, Z. S.
In: Genome, Vol. 60, No. 2, 2017, p. 183-192.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A semi-nested real-time PCR method to detect low chimerism percentage in small quantity of hematopoietic stem cell transplant DNA samples
AU - Aloisio, Michelangelo
AU - Bortot, Barbara
AU - Gandin, Ilaria
AU - Severini, Giovanni Maria
AU - Athanasakis, Emmanouil
AU - Xu, Z. S.
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
KW - Allogeneic HSCT
KW - Chimerism
KW - Semi-nested real-time PCR
KW - Sybr Green
UR - http://www.scopus.com/inward/record.url?scp=85011277124&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85011277124&partnerID=8YFLogxK
U2 - 10.1139/gen-2016-0092
DO - 10.1139/gen-2016-0092
M3 - Article
C2 - 28092167
AN - SCOPUS:85011277124
VL - 60
SP - 183
EP - 192
JO - Genome / National Research Council Canada = Genome / Conseil national de recherches Canada
JF - Genome / National Research Council Canada = Genome / Conseil national de recherches Canada
SN - 0831-2796
IS - 2
ER -