Duplex Polymerase Chain Reaction Quantification of Human Cells in a Murine Background

Abstract

Studies of the regenerative potential of human stem cells commonly involve their transplantation into immune-deficient mice or in vitro coculture with mouse cells. The optimal use of such models requires the detection and quantification of relatively low numbers of human cells in a murine background. We report here a duplex polymerase chain reaction (PCR) approach involving the coamplification of human-and mouse-specific repetitive sequences. The determination of product ratios compensates against variations in sample quality and enables quantitation from >50% down to 0.01% human-in-mouse from a single reaction. Product ratios are determined by standard electrophoresis of end-stage PCR reactions followed by image analysis techniques using freely available software, with no requirement for real-time PCR. The approach has been used to analyze tissue from mice transplanted with human cells and cocultures between differentiating mouse embryonal stem cells and human umbilical cord blood cells