Open Access

Methodology for detecting trace amounts of microchimeric DNA from peripheral murine white blood cells by real-time PCR

  • Carol M. Artlett1,
  • C. Gennaro Dito1 and
  • Paul J. Christner1Email author
Biological Procedures Online5:10103

DOI: 10.1251/bpo51

Received: 17 January 2003

Accepted: 9 March 2003

Abstract

Real-time PCR methodology can successfully quantitate microchimeric cell populations at a concentration of 100 microchimeric cells/100,000 host cells; however, it has not been successful in quantitating DNA from trace numbers of microchimeric white blood cells which we reported are present in murine peripheral blood at a concentration as low as 2/100,000 host cells. We report methodology using primers for a portion of the H2-kb murine histocompatibility sequence, specific for the C57BL/6J mouse. When these primers were used in the presence of 11,000 µM primer, a 20-fold increase in the median manufacturer’s recommended concentration, the assay could be optimized to detect 34 pg of C57BL/6J DNA in a background of 2.5 µg of carrier BALB/cJ DNA (1/100,000). These conditions resulted in a detection limit half as sensitive as that found when no carrier DNA was present.

Indexing terms

Polymerase Chain Reaction DNA Mice T-Lymphocytes leukocytes

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