Methodology for Detecting Trace Amounts of Microchimeric DNA from Peripheral Murine White Blood Cells by Real-Time PCR

• Main Authors: Artlett Carol M., Dito C. Gennaro, Christner Paul J.
• Format: Article
• Language: English
• Published: BMC 2003-01-01
• Series: Biological Procedures Online
• Subjects: Polymerase Chain Reaction; DNA; Mice; T-Lymphocytes; leukocytes
• Online Access: http://www.biologicalprocedures.com/bpo/arts/1/51/m51.htm
• ISSN: 1480-9222

<p>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^b murine histocompatibility sequence, specific for the C57BL/6J mouse. When these primers were used in the presence of 11,000 &mgr;M primer, a 20-fold increase in the median manufacturer&rsquo;s recommended concentration, the assay could be optimized to detect 34 pg of C57BL/6J DNA in a background of 2.5 &mgr;g 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.