Myeloid Leukemia

Diagnosis of CBFB-MYH11-Positive AML 173
4. Notes
1. All solutions and buffers (except for mineral oil and DMSO) used in PCR must
be stored at –20°C to avoid loss of quality that may result in inefficient PCR.
Mineral oil and DMSO should be stored at room temperature. Before use of frozen
material, thaw the required ingredients on ice. Materials used for agarose gel
electrophoresis can be stored at room temperature.
2. All steps should be carried out on ice to avoid loss of quality of PCR ingredients,
which may cause inefficient amplification.
3. The amount of PCR mix should be multiplied according the number of samples
that need to be analyzed (include mix for two additional samples because of loss
of mix during pipetting). Perform all analyses in duplicate.
4. Add mineral oil to avoid evaporation of the PCR mix during cycling when a PCR
machine without heated lid is used.
5. The described PCR cycling conditions were optimized using a DNA Thermal
Cycler 480 from Perkin Elmer. Because a large variation in PCR machines with
different performances exists, it may be necessary to adjust the annealing temperature
if the required sensitivity is not obtained or when nonspecific amplification
is observed. If adequate sensitivity is not obtained, the annealing temperature
is probably too high and should be lowered stepwise by 2°C until the desired
sensitivity is obtained. Conversely, if background or nonspecific amplification is
observed in controls negative for CBFB-MYH11, the annealing temperature
should be increased stepwise by 2°C.
6. The quality of RNA can be checked by regular agarose gel electrophoresis. Take
1 µg RNA in 10µL H2O and add 2 µL of 6X DNA loading buffer containing 5%
SDS. Run the samples in a fresh 1.5% agarose gel (0.5X TBE), which may also
be used for PCR product electrophoresis. Check for the presence of 18S and 28S
ribosomal RNA bands that, compared to DNA fragments, run at sizes of respectively
1.8 and 4.8 Kb. The presence of SDS in the DNA loading buffer will prevent
RNA degradation.
7. We have observed an average Ct value of 26.4 for PBGD when analyzing 50 ng
of cDNA of over 100 different patient samples (using a fixed threshold at 0.05).
Because degraded RNA can yield PBGD Ct values of 29, we advise setting the
maximal PBGD Ct value for individual patient samples at 28.4 (∆Ct of 2 relative
to the average, equaling a fourfold less efficient amplification compared to the
average value).
8. The quantitative range of a real-time PCR assay is determined by the lowest dilution
in the standard curve that shows a positive result in duplicate (∆Ct < 2).
Below this dilution, quantification is unreliable. Positive samples below this dilution
may be reported as weakly positive. For both the MYH11 and CBFB-
MYH11 qPCR, a sensitivity of 1 in 10,000 should be obtained.
9. The quality of real-time PCRs is determined using the calibration curve. The
slope of the calibration curve should lie between –2.8 and –3.8, a predefined
maximum Ct should be obtained for the undiluted sample of the calibration curve
at a fixed threshold, and negative controls should be negative.