Myeloid Leukemia

WT1 Expression in AML and MDS 209
2. The reactions can be carried out on alternative real-time detection systems, and
will yield comparable results that maintain the relationships between samples but
with different absolute values.
3. The collection of samples should be optimized in order to minimize the time
between sample acquisition and processing, as it has been demonstrated that the
stability of transcripts (including WT1) may be altered over time and with exposure
to different temperatures. Sodium citrate and EDTA are the optimal anticoagulants
for PB and BM, respectively, in order to avoid any interference with the
RQ-PCR procedure.
4. Use 1 mL of RNAwiz for a maximum of 10 7 cells.
5. Samples homogenized in RNAwiz can be stored at –20°C or –80°C for up to 1 mo.
6. The chloroform should not contain isoamylalchohol (IAA).
7. cDNA products can be stored at –20°C or –80°C for up to 1 yr.
8. It is mandatory to amplify a control gene so that impaired amplification of WT1
can be recognized by a corresponding reduction in the quantity of the control
gene that is detected. Variation in amplification could reflect variations in RNA
quality, quantity, and/or cDNA synthesis efficiency. Thus, quantification of ABL
expression is used to identify poor-quality samples, based on reference values
observed in a large number of fresh samples. The choice of ABL was decided by
a European network, because ABL expression is stable and constant in normal
and leukemic cells and in different cell types.
9. Alternative primer sets for WT1 assessment are present in the published literature.
This specific set possesses the advantage that it does not amplify genomic
DNA. It has been designed to avoid the two splicing regions present in exon 5
and 9, and the exon regions that are most frequently mutated in AML patients.
10. For WT1 quantitation in BCR-ABL-positive leukemias, it is more appropriate to
use GUS as the control gene instead of ABL, because the ABL primers will also
amplify the BRC-ABL fusion transcripts derived from the rearranged ABL allele.
That would result in an increased value for ABL, thereby altering the WT1/ABL
ratio, and consequently the WT1 value would be underestimated.
11. If one out of three amplified replicate samples shows a difference of more than 1
Ct with respect to the other two, we regard this as due to operator error, and the
discrepant replicate should be discarded. This rule applies for samples with a
WT1 Ct value below 40. For Ct values above 40, the amount of transcript is so
low that small differences in copy number may produce a large difference in Ct.
Accordingly, for samples with Ct values >40, we use the mean value of the three
replicates even when the Ct values for a specific sample differ by 2 or 3. In these
cases, discard only the replicates with a Ct value >50. For very low WT1 levels
(such as those present in normal PB samples), only one well out of three may give
a positive result; the final value is then represented by the single value obtained.
12. The no-template control is a well that contains the reaction mixture without cDNA.
13. Although test samples are assayed in triplicate, each plasmid dilution is loaded
in duplicate because they are very stable and the Ct values obtained are very
reproducible.