Myeloid Leukemia

Diagnosis of PML-RARA-Positive APL 119
2. 2.0 M Na acetate, pH 4.0.
3. Acid phenol, pH 4.3.
4. Chloroform-isoamyl alcohol, 49:1.
5. Isopropanol.
6. 75% ethanol kept at –20°C.
2.2. cDNA Synthesis
1. Random hexamers, 5 µM.
2. Diethylpyrocarbonate (DEPC)-treated water or double-distilled water for clinical
use.
3. 5X RT buffer: 20 mM Tris-HCl, 50 mM KCl, pH 8.3.
4. dNTP mixture of 10 mM each nucleotide.
5. 0.1 M dithiothreitol (DTT).
6. RNase inhibitor (40 U/µL).
7. Reverse transcriptase enzyme: Superscript II 200 U/µL (Invitrogen, Carlsbad,
CA).
2.3. Polymerase Chain Reaction
2.3.1. First-Round PCR Mixture
1. 0.2-mL plastic tube with cap.
2. 16.4 µL of water.
3. 2.5 µL of 10X reaction buffer: 200 mM Tris-HCl (pH 8.4), 500 mM KCl, final
concentration 1X.
4. 1.5 µL of 25 mM MgCl 2, final concentration 1.5 mM.
5. 0.5 µL of dNTP mix (10 mM each nucleotide), final concentration 0.2 mM.
6. 1.0 µL of 10 µM PML-A1 primer for bcr1/2 or PML-A2 primer for bcr3, final
concentration 0.4 µM.
7. 1.0 µL of 10 µM RARA-B primer (for both breakpoints), final concentration 0.4 µM
(see Table 1 for sequences).
8. 0.1 µL of Taq DNA polymerase (5 U/µL), final concentration 0.5 U/25 µL.
9. 2.0 µL of cDNA (100 ng of RNA equivalent).
Final reaction volume 25 µL.
2.3.2. Nested PCR Mixture
1. 17.4 µL of water.
2. 1.0 µL of first-round PCR product (in place of cDNA).
3. Use the same final volume and the same reagents as for the first-round PCR, with
the following exception: use PML-C1 for bcr1/2, or PML-C2 for bcr3 as the
forward primers, and use RARA-C as the reverse primer for both breakpoints.
2.4. Gel Electrophoresis
1. 5X Tris-borate electrophoresis buffer (TBE): 54 g Tris-base, 27.55 g boric acid,
20 mL of 0.5 M ethylenediamine tetraacetic acid (EDTA). Make up to 1 L and
store at room temperature.