Myeloid Leukemia

Duplexed QZyme RT-PCR for APL Analysis 141
1. Set up duplex QZyme PCR PML-RARα-BCR reactions as described under Subheading
3.4.
2. At Subheading 3.4., step 3, the normal calibrator curve (consisting of five calibrators)
should be extended above the highest calibrator and below the lowest
calibrator, normally assessed, to produce a total of seven calibrators. Five replicates
of each of these seven calibrators should be measured to allow precision
and accuracy to be calculated.
3. Thermocycle the reactions and export the data (Subheading 3.4., steps 8–10).
The standard curve should be generated from only the middle five calibrators
(those known to produce a linear relationship between Ct and copy number from
previous experiments).
4. For each of the seven calibrators, calculate the following values (see Note 20):
CV Ct (%) = (100 × SD Ct)/average Ct
CV copy # (%) = (100 × SD copy #)/average copy #
Accuracy (%) = 100 × (average measured copy #)/(known copy #)
5. The upper limit of quantitation (ULOQ) is the highest concentration of calibrator
that meets the acceptance criteria of CV CT < 2%, CV copy # < 25%, and accuracy
within 70% to 130% of the known calibrator value.
6. The lower limit of quantitation (LLOQ) is the lowest concentration that meets
the acceptance criteria of CV CT < 3%, CV copy # < 30%, and accuracy within 70%
to 130% of the known calibrator value (see Note 20).
3.6.3. Reproducibility
Reproducibility is a measure of the repeatability of results performed within
one run (intra-assay) and between runs (inter-assay). In our experience with
the ABI PRISM 7700, the variation between sample wells on the 96-well block
is greater than the variation seen for the same well between runs. This variation
is also dependent on the amount of signal generated, and thus on the amount of
template. Intra- and inter-assay variation are measured by a set of three experiments,
as follows:
1. Inter-assay variation is determined by the results of the t-tests performed on a
series of controls analyzed over three experiments. Set up Duplex QZyme PCR
PML-RARα-BCR reactions as described under Subheading 3.4.
2. At Subheading 3.4., step 3, the calibrator curve should be run in duplicate.
3. Include five replicates of three control samples: “high control” with 100,000 copies
of PML-RARα plasmid in 500 ng total RNA, “medium control” with 20,000
copies of PML-RARα plasmid in 500 ng total RNA, and “low control” with 4000
copies of PML-RARα plasmid in 500 ng total RNA. All calibrators and the five
replicates of the three controls should be assessed in the same wells for each of
the three experiments.