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54 Picard, Silvy, and Gabert<br />
Few studies have focused on the definition of control genes. A collaborative<br />
action was undertaken within the EAC program involving 6 laboratories among<br />
26 from 10 member countries. This program defined and standardized RQ-<br />
PCR protocols suitable for measuring the levels of FG in leukemia with special<br />
reference to the selection and optimization of CGs (31). This multicenter retrospective<br />
study on over 250 acute and chronic leukemia samples obtained at<br />
diagnosis and with an identified FG confirmed that three CGs (ABL, β-glucuronidase<br />
[GUS], and β-2-microglobulin [B2M]) had stable expression within<br />
the different samples. However, only ABL transcript expression did not differ<br />
significantly between normal and leukemic samples at diagnosis, and therefore<br />
the EAC proposes to use ABL as the CG for RQ-PCR-based diagnosis and<br />
MRD detection in leukemic patients.<br />
It should be noted that Roche Diagnostics and Applied Biosystems market<br />
different kits that evaluate the expression of a variety of housekeeping genes.<br />
3.4.2. Quantification<br />
RQ-PCR can be subdivided into two basic categories: absolute and relative<br />
quantification.<br />
3.4.2.1. ABSOLUTE QUANTIFICATION<br />
Absolute quantification utilizes a standard curve derived from known<br />
amounts of a calibrator. If quantitation is normalized to an endogenous control,<br />
a second standard curve for the endogenous reference must also be prepared.<br />
Then, the target amount is divided by the endogenous reference amount to<br />
obtain a normalized target value. The calibration curves used in absolute quantification<br />
can be based on known concentrations of DNA standard molecules<br />
(recombinant plasmid DNA), genomic DNA, RT-PCR products, or commercially<br />
synthesized large oligonucleotides. Recombinant plasmid DNA dilutions<br />
in Escherichia coli 16S and 23S rRNA (20 ng/µL) are most widely used. This<br />
allows the determination of absolute gene-expression levels in unknown<br />
samples. Plasmids are robust and helpful for standardization and reproducibility<br />
of results within multicenter studies. Because the use of plasmids leads to<br />
highly reproducible results, the threshold can be fixed, allowing a direct comparison<br />
of Ct values or copy numbers (CN) for the same sample in different<br />
laboratories. Furthermore, their storage is very easy (they are stable for several<br />
years at –20°C). However, there are limitations to the use of plasmids.<br />
First, they allow validation of the PCR step but not of the reverse transcriptase<br />
step. Second, contamination risk is relatively important and requires a separate<br />
laboratory for handling plasmids, or the use of degradable dU-based DNA templates.<br />
Furthermore, plasmid design and construction is a relatively long process<br />
(involving standard synthesis, purification, cloning, transformation,
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