Myeloid Leukemia

48 Picard, Silvy, and Gabert
Some companies produce probes (Applied Biosystems, Genset Corp,
Eurogentec, Operon Technologies, Sigma-Genosys) and some offer design and
synthesis (MWG, Proligo). Some software applications are available for the
design of primers and probes. Among these, Primer Express (Applied
Biosystems) allows the design of dual labeled probes, and the LightCycler
Probe Design software allows the design of hybridization probes. Additional
primer programs for real-time PCR primer and probe design are available
online (http://www.molecular-beacons.org; http://www.genome.wi.mit.edu/
cgi-bin/primer/primer3_www.cgi; http://alces.med.umn.edu/webprimer.html).
Many sequences of primers and probes for fusion gene transcripts or control
genes are relatively comparable between the various AML studies. This is
explained principally by the use of the same software for their design. It should
be noted that certain published sequences (Table 2) are false or comprise various
errors, whereas the probe and primer sequences we have reported within
the EAC program can be used immediately (30,31).
3. At the Bench
3.1. Preparation of a Run
Depending on the RQ-PCR instrument used, real-time PCR is carried out in
96-well reaction plates, in 32 glass capillaries, or in single tubes. However,
general considerations are identical in all cases. The PCR mix can be prepared
at room temperature, because the DNA polymerase is inactivated either by
chemical modification or by an antibody. For clinical evaluation, the use of a
master mix (containing buffer, MgCl2, dNTPs, SybrGreen (if used), and
enzyme) is preferred to minimize handling and subsequent inter-assay variations.
The optimal probe concentration is 50–200 nM (ideally 100 nM), and the
primer concentration is 100–900 nM (ideally 300 nM for reverse and forward
primers). These variations in concentration are generally dependent on the company
that synthesizes the primers and probes. PCR amplifications are performed
in a total volume that varies depending on the instrument used (for
example, 50 µL or 25 µL for 7700/7000 ABI, 20µL for LightCycler). Patient
samples are ideally analyzed in duplicate for control genes and in triplicate for
fusion gene transcripts. However, when using low-throughput instruments, a
single control gene analysis and a duplicate for fusion genes can be performed.
PCR conditions are 10 min at 94°C followed by a total of 40–50 two-temperature
cycles (15 s at 94°C and 1min at 60°C).
For absolute quantification using plasmid standards, a calibration curve for
each transcript is generated. For control gene amplification, three plasmid
dilutions between 100,000 and 1000 copies are sufficient, because for lower
copy number, the quantity/quality of sample is low, leading to either a loss of