Quantitative polymerase chain reaction-based assay with fluorogenic Y-chromosome specific probes to measure bone marrow chimerism in mice

Abstract

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.