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Quantitative polymerase chain reaction-based assay with fluorogenic Y-chromosome specific probes to measure bone
Stefan Oliver Peters1, Kathrin Bauermeister, Jan Philipp Simon
1Laboratory of Experimental Stem Cell Biology, Division of Hematology, Medical Department I, University of Luebeck, Luebeck, Germany. Stefan.Peters@medinf.mu-luebeck.de
Unlabelled:
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.
Materials And 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.
Conclusion:
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.