Myeloid Leukemia

Real-Time RT-PCR Strategies 51
AML-ETO, and CBFB-MYH11, which were based on Taqman chemistry, were
designed during the first meeting in Rotterdam, and were then optimized during
phase II, for a definitive selection during phase III, and overall testing in
phase IV (30). For PML-RARA transcripts, the three breakpoint regions bcr1,
bcr2, and bcr3 can be detected by the different sets designed by the EAC program.
Similarly, three different sets were developed for the most frequently
occurring CBFB-MYH11 fusion gene transcripts—types A, D, and E.
This EAC protocol can be transferred directly with no adaptation onto different
instruments, like the ABI 7000/7700, Light Cycler, Smart Cycler, Rotor-
Gene, iCycler, and MX4000 (28a).
3.2.2. Several Kits Are Commercially Available
AME Biosciences and Roboscreen have developed a quantification kit:
Amplitect PML-RARA cDNA quantification module, which detects breakpoint
region bcr1. Its use requires the housekeeping gene quantification module:
GAPDH, 18S rRNA, or ABL cDNA. Each lot of amplitect consists of FG control
DNA at different concentrations per eight-well control strip, and FG real
time reagent mix (primers and probe). The TaqMan chemistry is used with
ABI 7000/7700, GeneAmp 5700, iCycler, Smart Cycler, and Rotor-Gene.
Roche Diagnostics promotes a number of quantification kits for the Light-
Cycler:
• LightCycler Inv(16) quantification kit.
• LightCycler t(8;21) quantification kit.
• LightCycler t(15;17) quantification kit.
These kits are specifically adapted for PCR in glass capillaries using the
LightCycler instrument. Each lot provides all reagents required for the quantification
of the FG RNA in a two-step procedure (RT and PCR). The amplicon
is detected by using a specific pair of hybridization probes. Results are
expressed as the ratio of FG/G6PDH in the sample, relative to the ratio of FG/
G6PDH in the calibrator. For analysis, a calibrator provided with the parameter-specific
LightCycler quantification kits is included in each LightCycler
run. For inv(16) quantification, the LightCycler Inv(16) quantification kit and
the EAC protocol have identical detection sensitivities (unpublished data).
3.3. Quality Controls
RQ-PCR has become an important tool in research as well as in clinical
diagnostics. However, the RT-PCR reaction is fraught with hazards (32) and is
characterized by significant variation and nonreproducibility between different
laboratories, even with identical samples (33,34). Clearly, there is an urgent
requirement for stringent quality control and standardization of sample acqui-