Quantification of human cells in NOD/SCID mice by duplex real-time polymerase-chain reaction.

BACKGROUND AND OBJECTIVES The aim of this study was the development of a fast and reliable polymerase chain reaction (PCR) assay which quantifies the proportion of human cells in immunodeficient chimeric mice, for example transplanted with human hematopoietic stem cells. DESIGN AND METHODS We developed a TaqMan chemistry-based, real-time duplex PCR assay to quantify human and murine DNA in a single-tube reaction in parallel (HUmu PCR). Two independent sets of primers and exonuclease probes, located in the tumor necrosis factor-a gene of both species, were selected to amplify specifically human and murine genomic DNA. Serial dilutions of defined numbers of human cells in mouse cells served to construct calibration curves. The test was applied to NOD/SCID mice transplanted with CD34(+) cells isolated from human cord blood and compared to FACS analysis. RESULTS Analysis of DNA from human cells diluted stepwise into a fixed number of murine cells - and vice versa - led to calibration curves with good correlation for human and murine cells (r(2)>0.99) with a detection limit of 2% human cells. Results obtained with the HUmu PCR paralleled those of FACS analysis. However, in contrast to FACS analysis, which requires fresh single cell suspensions, the HUmu PCR can be carried out on already stored samples, even from solid organs and, moreover, the quantity of material required for analysis is very low. INTERPRETATION AND CONCLUSIONS The HUmu PCR presented here is the first real-time PCR assay for simultaneous quantification of human and murine cells. It is extremely fast, accurate and is an interesting alternative method for quantifying the proportion of human DNA in organs of chimeric mice.