Myeloid Leukemia

WT1 Expression in AML and MDS 205
The fluorescent Taqman probes are labeled with 6-carboxy-fluorescein
phosphoramide (FAM) as reporter dye at the 5�-end, and the quencher dye is 6carboxytetramethylrhodamine
(TAMRA) at the 3�-terminus.
The PCR procedure starts with:
a. 2 min at 50°C to activate the UNG enzyme, then
b. 10 min at 95°C to inactivate the UNG enzyme and to provide a “hot start” for
activating the AmpliTaq polymerase.
c. Subsequently, 50 cycles of denaturation at 95°C for 15 s followed by annealing/extension
at 60°C for 60 s.
6. Perform all sample analysis in triplicate; where the results show a discrepancy of
more than 1 threshold cycle (Ct) in one of the wells, that replicate must be
excluded (see Note 11).
3.4. Generation of Plasmid Standard Curves
1. To construct a WT1 plasmid for the standard curve in the RQ-PCR assay, add the
following primers and reagents in a final volume of 50 µL to amplify a WT1 RT-
PCR product:
forward primer: exon 7 -5�-GGCATCTGAGACCAGTGAGAA-3�
reverse primer: exon 10 5�-GGACTAATTCATCGACCGGG-3�
a. PCR buffer 1X:10 mM Tris-HCl, 50 mM KCl (pH 8.3)
b. MgCl 2: 2.5 mM (final concentration)
c. dNTP: 200 µM (final concentration)
d. 400 nM of each primer (final concentration)
e. Taq enzyme: 1 U
f. 3 µL of cDNA product
Use the following thermal cycler temperatures and time conditions:
a. Initial melting 95°C for 30 s
b. 35 PCR cycles at the following conditions: 94°C for 30 s, 65°C for 1 min,
72°C for 1 min.
2. Clone the PCR products into the PCR II TOPO vector (Invitrogen, Groningen,
The Netherlands) according to the manufacturer’s instructions.
3. Sequence the selected plasmid for confirmation of the insert, and extract the
plasmid DNA using the Qiafilter Plasmid MIDI kit (Qiagen, Courtaboeuf, France)
and quantify spectrophotometrically. The copy number for 1 µg is estimated
according to the molecular weight of the vector plus the insert.
4. Linearize 20 µg of plasmid with BamHI or HindIII restriction enzymes for 1 h at
37°C under agitation.
5. Dilute the digested plasmid in a solution of Tris 10 mM, EDTA 1 mM (pH 8),
containing 20 ng/mL of E. coli 16S and 23S rRNA (Roche).
6. Prepare five successive serial dilutions (20,000, 2000, 200, 20, and 2 copies/
µL). The corresponding standard curve generates a mean slope of –4.04 and an
intercept of 46.75 Ct. A mean Ct value of 26.4 should be obtained for 100,000
copies of the plasmid dilution (see Fig. 1). The actual values achieved (as in Fig.