Myeloid Leukemia

142 Mokany et al.
4. Thermocycle the reactions and export the data (Subheading 3.4., steps 8–10).
Repeat steps 1–4 twice to total three experiments with exactly the same reactions.
Use the same aliquot of calibrators and controls for all three experiments.
5. For the five replicates of each control within each experiment, calculate the following:
Average normalized copy #
SD normalized copy #
CV normalized copy # (%) = 100 × SD normalized copy # / average normalized copy #
6. Calculate the following ratios for the high control:
Experiment #1 Average normalized copy #/Experiment #2 Average normalized copy #
Experiment #1 Average normalized copy #/Experiment #3 Average normalized copy #
Experiment #2 Average normalized copy #/Experiment #3 Average normalized copy #
7. Repeat step 6 for the medium and low controls
8. Perform t-tests between the following values:
Replicates of high control normalized copy #: Experiment #1 vs Experiment #2
Replicates of high control normalized copy#: Experiment #1 vs Experiment #3
Replicates of High Control normalized copy#: Experiment #2 vs Experiment #3
9. Repeat step 8 for the medium and low controls.
10. If the difference in Average normalized copy # for a control from different experiments
does not reach statistical significance (p < 0.05) then there is no real difference
between the Average normalized copy# generated by the two experiments, and the ratio
is irrelevant. Statistically significant differences may not necessarily be significant
in the context of the assay. Real-time quantitative PCR assays have a
maximum sensitivity of twofold, and thus any ratio between experiments that
falls between 0.5 and 2 is generally acceptable. In our hands, the ratio between
experiments for the high and medium controls fall between 0.67 and 1.5 (a variation
of 50%). The low control shows greater variability, and we measure ratios of
between 0.56 and 1.8 (an 80% variation) (see Note 21).
11. Intra-assay variation is determined from the CV normalized copy # for each control. In
our experience, the CV normalized copy # for the high and medium controls should fall
below 25%, and that for the low control below 35%.
4. Notes
1. Most patients harbor either the L- or S-type, whereas approx 10% of patients
harbor the V-type. In V-type patients, the breakpoint usually occurs within exon