Duplex polymerase chain reaction quantification of human cells in a murine background.
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.
Pelz
Oliver
O
Laboratory of Molecular Medicine, Interdisiplinary Centre for Clinical Research, University of Leipzig Faculty of Medicine, Germany.
Wu
Minyao
M
Nikolova
Teodora
T
Kamprad
Manja
M
Ackermann
Manuela
M
Egger
Dietmar
D
Emmrich
Frank
F
Wobus
Anna M
AM
Cross
Michael
M
D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D018920:Coculture Techniques; D017931:DNA Primers / Q000737:chemistry; D004730:Endothelium, Vascular / Q000166:cytology; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D006023:Glycoproteins / Q000096:biosynthesis; D006801:Humans; D007091:Image Processing, Computer-Assisted; D051379:Mice; D010455:Peptides; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014470:Umbilical Cord / Q000166:cytology
D000071916:AC133 Antigen; D015703:Antigens, CD; D017931:DNA Primers; D006023:Glycoproteins; D010455:Peptides