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




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Biological Procedures Online

, Volume 5, Issue 1, pp 103–107

Received: 17 January 2003Revised: 05 March 2003Accepted: 09 March 2003DOI: 10.1251-bpo51

Cite this article as: Artlett, C.M., Dito, C.G. & Christner, P.J. Biol. Proced. Online 2003 5: 103. doi:10.1251-bpo51

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-k 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 termsPolymerase Chain Reaction DNA Mice T-Lymphocytes leukocytes Published: April 7, 2003

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Autor: Carol M. Artlett - C. Gennaro Dito - Paul J. Christner

Fuente: https://link.springer.com/







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