Myeloid Leukemia

56 Picard, Silvy, and Gabert
and is critically influenced by PCR reaction components. Constant amplification
efficiency in all samples being compared is one important criterion for
reliable comparison between samples. Small efficiency differences between
target and reference generate false expression ratios, and the true initial level
of mRNA may be over- or underestimated. Several mathematical methods that
determine real-time PCR efficiency have been described (42–45) and are compared
online (http://www.gene-quantification.info/). The equivalence of PCR
efficiency between test samples and calibrators should be validated in an independent
experiment.
Efficiency-corrected quantifications should be included in the setup and calculations
that are performed in relative quantification procedures. LightCycler
Relative Expression, Q-gene, Rest, and Rest-XL software applications allow
the evaluation of amplification efficiency plots. Q-gene, Rest, and Rest-XL
software are available directly on the Web and are applicable on different
instruments, whereas LightCycler Relative Expression is an application of
LightCycler that permits the use of one to two calibration wells for each series.
For example, the LightCycler kits are function tested using calibrator RNA
derived from an immortalized cell line for each FG expression and do not
require a standard curve. Instead, the amount of mRNA encoding the FG is
expressed as a ratio relative to a reference gene (G6PDH) in that particular
sample, and then compared to the FG:G6PDH ratio in the calibrator.
4. RQ-PCR Results
4.1. Expression of the Results
Based on the quantification of (1) CG expression in a patient’s follow-up
samples and (2) FG and CG expression in the patient’s diagnostic sample, two
methods are most commonly used for calculation of MRD results and experimental
sensitivity: the ∆∆Ct method and the normalized copy number (NCN)
method.
With the ∆∆Ct method, the results can be calculated using slopes and intercepts
derived from previously generated plasmid standard curves. Based on
the EAC results, average intercept and slope values of 40 and –3.4, respectively,
can be used for such calculations.
Alternatively, the NCN method requires inclusion of plasmid standards in
each quantitative PCR experiment. The NCN method has been most frequently
used for reporting MRD data in the literature. The levels of expression of fusion
transcripts in both patient and control samples are expressed as normalized
copy numbers by calculating the ratio of fusion gene transcript copy number
divided by the control gene transcript copy number, and adjusted as required
for ease of interpretation (e.g., multiplication by 100 or 1000). The NCN
method is robust and is the preferred method for standardization of results