Myeloid Leukemia

Duplexed QZyme RT-PCR for APL Analysis 137
Fig. 2. Amplification plots and calibration curves for single-tube duplex assay.
Examples of duplex assays for (A) PML-RARα L-type, (B) BCR control
8. Click on “thermal cycling” and program the following profile (see Note 14):
60 min at 55°C, 3 min at 95°C
10 cycles of 95°C for 15 s and 68°C for 40 s (–1°C/cycle),
40 cycles of 95°C for 15 s and 55°C for 40 s
9. Load plate, close the lid, and select “run.”
10. Save data after completion of run (see Note 15).
11. Analyze data from the ABI 7700 according to the guidelines from Applied
Biosystems (Note 16). For both PML-RARα assays, set the baseline and thresholds
at 3–12 and 0.2, respectively. For the BCR assays, set the baseline and threshold
at 3–15 and 0.07. Examples of an amplification plot and a standard curve
from the L-type duplex assay are given in Fig. 2 (see Note 17).
3.5. Relative Quantitation of Transcripts in Patient Samples
To determine the correct primer set to use in QZyme RT-PCR, it is recommended
that the PML-RARα isoform harbored by each patient be identified by
standard qualitative RT-PCR (see Chapter 7).
The single-tube QZyme RT-PCR method described in this chapter is not
only able to confirm the presence of the PML-RARα transcript in each patient,
but also estimate its relative abundance. The quantities of PML-RARα and BCR
transcripts, amplified simultaneously in a duplex reaction, are estimated from
each calibration curve (see Subheadings 3.1. and 3.2.). The relative abundance
of transcripts is then calculated by normalization of PML-RARα to the control
BCR transcripts, to account for variation in RNA concentration and quality,
according to the equations in Subheading 3.5., step 2). The accurate assessment
of RNA quality in each specimen, which can vary considerably, is essen-