Methodology for Detecting Trace Amounts of Microchimeric DNA from Peripheral Murine White Blood Cells by Real-Time PCR.
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 microM 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 microg 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.
Division of Rheumatology, Thomas Jefferson University. 233 S 10th Street, Room 509 B.L.S.B., Philadelphia, PA, 19107. USA.
Carol M.
CM
Artlett
C Gennaro
CG
Dito
Paul J.
PJ
Christner