Myeloid Leukemia

206 Cilloni, Gottardi, and Saglio
Fig. 1. The Wilms tumor gene (WT1) amplification plot and standard curve with 6carboxytetramethylrhodamine
(TAMRA) probe. In the amplification plot, the threshold
was set to a value of 0.1 and the baseline to between cycles 3 and 15. The standard
curve generates a slope of –3.975 and an intercept of 46.475. The mean threshold
cycle (Ct) values for each of the plasmid copy numbers obtained by serial dilutions of
the plasmid are indicated to the right of the figure.
1) may vary slightly from these theoretical values for several reasons, including
variability in the efficiency of the reaction and individual operator manual variability.
This should not cause inaccuracies in the final quantitation of copy numbers,
because the latter are normalized using the ABL gene.
7. To prepare the ABL control gene standard curve, use commercial ABL plasmid
(Ipsogen, Marseille, France).
8. Use three different serial dilutions (20,000, 2000, and 200 copies/µL). The
corresponding standard curve generates a mean slope of –3.64 and an intercept of 40
Ct. A mean Ct value of 21.78 should be obtained for the 100,000 copies of the
plasmid dilution (see Fig. 2). The actual values achieved may vary slightly from
these theoretical values, as discussed above.
9. Store the plasmid dilutions at –20°C. Aliquot the serial dilutions in order to avoid