Myeloid Leukemia

Real-Time RT-PCR Strategies 57
between various laboratories. This is crucial if RQ-PCR is to be used as a clinical
tool for AML patients. Indeed, as shown by nucleic acid quantification
technology in virology, the final goal is to define international units allowing
data comparisons of different therapeutic strategies.
Few authors in the research field use the ∆∆Ct relative quantification (relative
expression) approach (described previously, and in more detail in the
Applied Biosystems User’s Bulletin #2). The Ct values are normalized for an
endogenous reference control gene (∆Ct = Ct FG – Ct CG) and compared with a
calibrator (∆∆Ct = ∆Ct samples – ∆Ct calibrator). The calibrator can be a dilution of
a cell line, or the average Ct value of several bone marrow or blood samples
from healthy volunteers. The comparative ∆∆Ct method requires similar PCR
efficiency (more than 90%) for both target and control genes. Different efficiencies
may be observed between patients and standards; thus, a validation
experiment is required. cDNA from randomly selected patients and healthy
donor samples as well as from cell lines are serially diluted over a concentration
of 1:1 to 1:1000. All samples are analyzed in triplicate and the resulting
median used for further calculation. The resulting Ct values are plotted against
the log 10 concentration of total RNA. The slope of the fitted line is then determined.
A slope of less than 0.1 indicates that the efficiency of amplification of
the target or control genes between the various samples is equivalent.
In this area, it is worth noting that the LightCycler Relative Quantification
software provides a tool for fully automated calculation of calibrator-normalized
and PCR efficiency-corrected relative quantification of mRNA expression
levels or gene dosage values. The great advantage of this system is that
standards are not required in each LightCycler analysis run, because stored
files containing fitted coefficients can be used instead. To generate analysis
parameters for storage and use in subsequent assays, plasmid or cell line dilutions
for both target and control genes are performed several times. The resultant
Ct values are plotted against the log of the concentration, and two different
regression fits are performed. A linear fit is calculated using LightCycler Data
Analysis (LCDA) software, and a nonlinear fit is performed by exporting
LCDA data into the coefficients module of the Relative Quantification software.
The automatically calculated fit coefficient is stored in a file for subsequent
analysis. This software provides a method to compensate for the observed
“low concentration shift” of the Ct values. Relative concentrations of target
and reference genes of each sample and calibrator are calculated automatically
from this curve.
The calibrator can be a cell line or plasmid dilutions at known concentration.
For example, for AML1-ETO detection, Kasumi cell RNA diluted to 10 –3
or plasmid concentration of 1000 copies can be used. NB4 cells and ME-1 cells
can be similarly used for PML-RARA bcr1 and CBFB-MYH11 type A, respec-