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 f ast and reliable polymerase chain reaction (PCR) assay which quantifies the proportion of human cells in immunodeficient chimeric mice, for Example tr ansplanted 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 pa rallel (HUmu PCR). Two independent sets of primers and exonuclease probes, located in the tumor necrosis factor-alpha gene of both species, were selec ted to amplify specifically human and murine genomic DNA. Serial dilutions of defined numbers of human cells in mouse cells served to construct calibr ation 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 num ber 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 o f 2% human cells, Results obtained with the HUmu PCR paralleled those of FA CS analysis. However, in contrast to FACS analysis, which requires fresh si ngle cell suspensions, the HUmu PCR can be carried out on already stored sa mples, even from solid organs and, moreover, the quantity of material requi red for analysis is very low. Interpretation and Conclusions. The HUmu PCR presented here is the first re al-time PCR assay for simultaneous quantification of human and murine cells . It is extremely fast, accurate and is an interesting alternative method f or quantifying the proportion of human DNA in organs of chimeric mice. (C) 2001, Ferrata Storti Foundation.