Myeloid Leukemia

204 Cilloni, Gottardi, and Saglio
3.2. RT Reaction
1. Use 1 µg of total RNA and incubate at 70°C for 10 min.
2. Cool on ice and add the following reagents plus H 2O to a final theoretical volume
of 20 µL. Depending on the commercial availability of a stock solution of
random hexamers at sufficiently high concentration, the exact final volume of
the reagent mix may be as much as 21.8 µL:
a. RT buffer 1X: 10 mM Tris-HCl, 50 mM KCl (pH 8.3).
b. MgCl 2: 5 mM (final concentration).
c. DTT: 10 mM (final concentration).
d. dNTP: 1 mM each (final concentration).
e. Random hexamers: 25 µM (final concentration).
f. RNAse inhibitor: 20 U.
g. Reverse transcriptase enzyme 100 U.
3. Use the following thermal cycler temperatures and time conditions:
a. 20°C for 10 min.
b. 42°C for 45 min.
c. 99°C for 3 min.
d. 4°C at the end of the RT step.
4. After the RT reaction, add RNase-free water to a final volume of 50 µL (see Note 7).
3.3. Real-Time Quantitative RT-PCR Step
1. All the real-time quantitative RT-PCR (RQ-PCR) reactions are performed on a
7700 ABI platform (see Note 2).
2. Perform each run using a specific set of primers and probe for WT1 and ABL (see
Note 8).
3. Perform the PCR reaction for WT1 and ABL as follows:
a. Use 5 µL of diluted cDNA (corresponding to approx 100 ng of starting RNA).
b. 12.5 µL of TaqMan Universal PCR Master Mix (Applera).
c. 300 nM of each primer.
d. 200 nM of the probe.
e. Add sterilized water to reach a final volume of 25 µL.
4. The RQ-PCR primers and probe for WT1 are (see Note 9):
forward primer (located on exon 7):
5�- CAGGCTGCAATAAGAGATATTTTAAGCT-3�
reverse primer (located on exon 8):
5�-GAAGTCACACTGGTATGGTTTCTCA-3�
TaqMan probe (located on exon 7):
5�-CTTACAGATGCACAGCAGGAAGCACACTG-3�.
5. The RQ-PCR primers and probe for ABL (see Note 10) are:
forward primer 5�-TGGAGATAACACTCTAAGCATAACTAAAGGT-3�
reverse primer 5�-GATGTAGTTGCTTGGGACCCA-3�
TaqMan probe 5�-CCATTTTTGGTTTGGGCTTCACACCATT-3�