Quantitative polymerase chain reaction-based assay with fluorogenic Y-chromosome specific probes to measure bone marrow chimerism in mice.
A quantitative duplex polymerase chain reaction (PCR) method using the LightCycler detection system was developed to generate a reproducible and convenient method to quantitatively measure chimeric states in murine transplantational models using male and female BALB/c mice.


METHODS
DNA mixtures isolated from male and female murine BALB/c bone marrow cells were analyzed with Y-chromosome and control autosomal GAPDH specific primers by either monoplex or duplex real-time quantitative PCR using the LightCycler detection system to determine chimeric states.


RESULTS
High specific Y-chromosome and control autosomal GAPDH primers gave a detection sensitivity of approximately 0.01% using LightCycler PCR. Analysis of standard curve distribution of diluted DNA samples accurately matched the proportional dilutions. Y6 primers run on male DNA samples against a female background showed amplification slope initiations reflecting the amounts of male DNA contained in these mixtures. Mixed samples run with GAPDH primers showed amplification corresponding to 100% DNA amounts of limiting dilution runs which were used as controls. Calculated engraftment generated by monoplex PCR lacked reproducibility. Quantitation using duplex PCR using specific detection probes for both Y-chromosome Y6 and autosomal GAPDH primer amplicons allowed precise and reproducible calculations of engraftment levels.


CONCLUSIONS
Duplex PCR using specific detection probes for Y-chromosome Y6 and autosomal GAPDH primer amplicons allows for rapid, precise and specific engraftment determination after transplantation of male BALB/c marrow cells into female BALB/c recipients.
Peters
Stefan Oliver
SO
Laboratory of Experimental Stem Cell Biology, Division of Hematology, Medical Department I, University of Luebeck, Luebeck, Germany. Stefan.Peters@medinf.mu-luebeck.de
Bauermeister
Kathrin
K
Simon
Jan Philipp
JP
Branke
Biggi
B
Wagner
Thomas
T
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016026:Bone Marrow Transplantation; D005260:Female; D005456:Fluorescent Dyes; D006085:Graft Survival; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction / Q000379:methods; D011237:Predictive Value of Tests; D018183:Transplantation Chimera; D014998:Y Chromosome
D005456:Fluorescent Dyes