A “Real Time” PCR Assay to Detect Transplanted Human Liver Cells in RAG-1-/- Mice
Abstract:Xenotransplantation of human liver cells is an expanding field in need of new and precise quantitative techniques. “Real time” PCR is a sensitive and accurate method of quantifying picogram quantities of DNA. We used “real time” PCR with primers complementary to the human α-1-antitrypsin gene to assess the efficiency of engraftment of human liver cells transplanted into immunotolerant RAG-1-/- mice. Standard curves were created by mixing known proportions of human and mouse cells. There was a linear relationship between the PCR cycle at which DNA was amplified [threshold cycle (CT)] and the percent human cells (linear regression, p < 0.00009). Results were reliable, with a maximum 1.27-fold variation in the slopes of repeated standard curves. Linearity was maintained from 100% to as low as 0.01%. Therefore, 1 in 10,000 mouse cells could be detected in a 100 ng DNA sample. We measured the percent engraftment of human liver cells transplanted into the spleen of RAG-1-/- mice. By “real time” PCR assay, 0.23% human cells could be detected at 1 day after human cell transplantation. These results show that “real time” PCR assay is highly sensitive, reproducible, and accurate for detecting human cells in xenotransplanted mice.
Document Type: Research Article
Affiliations: 1: *University of Chicago, Department of Pediatrics, University of Chicago Children's Hospital, Chicago, IL 60637 2: †Northwestern University, Department of Pediatrics, Children's Memorial Hospital, Chicago, IL 60614
Publication date: 2001-02-01
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