Myeloid Leukemia

84 Branford and Hughes
5. Pipet 22.5 µL of the first-step master mix and 23 µL of the second-step master
mix into 0.2-mL tubes. Store the second-step tubes at 4°C until completion of the
first-step PCR.
6. In a separate laboratory, add 2.5 µL of the cDNA to the first-step mix, mix thoroughly,
and centrifuge briefly to collect the mix at the bottom of the tube.
7. Transfer the reactions to the thermal cycler and amplify with the following conditions:
a. 1 cycle at 95°C for 10 min.
b. 30 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 30 min.
c. 1 cycle at 72°C for 7 min.
8. The following steps are performed in a separate area from the PCR master mix
setup and the cDNA addition. After completion of the first-step PCR, dilute the
PCR products 1 in 100 in sterile water. Mix the tubes thoroughly and centrifuge
briefly to collect the mix at the bottom of the tube.
9. Add 2 µL of the 1 in 100 dilution to the second-step mix. Mix the tubes thoroughly
and centrifuge briefly to collect the mix at the bottom of the tube.
10. Transfer the reactions to the thermal cycler and amplify with the same conditions
as under Subheading 3.7.7.
11. After completion of the PCR, assess the size of each amplification product by
electrophoresis of 5 µL of the PCR product mixed with 1 µL gel loading buffer
on a 2.0% agarose gel, using the Puc19 size marker.
12. The size of the first-step b2a2 BCR-ABL product is 220 bp, and the b3a2 BCR-
ABL product is 295 bp. The size of the second-step b2a2 BCR-ABL product is 142
bp, and the b3a2 BCR-ABL product is 217 bp. An unusual PCR artifact fragment
may be present when the patient has both the b2a2 and b3a2 BCR-ABL transcripts
(see Note 6).
13. The sensitivity of the second-step PCR is one K562 cell diluted in 10 6 BCR-ABL
negative cells.
4. Notes
1. Measures are in place throughout every step to prevent cross-contamination of
plasmid or patient samples as well as contamination with PCR product. The
majority of patients undergoing analysis have minimal residual disease or undetectable
BCR-ABL levels, and it is therefore essential for patient management
that false-positive results do not occur. The main area of concern is in the preparation
and dilution of high-copy-number plasmids. This is done in a separate
laboratory from the RNA extraction and PCR setup laboratories, using separate
dedicated equipment. Other measures include the use of sterile barrier tips, UVirradiated
pipets, RNAse- and DNAse-free tubes, and dedicated equipment for
each procedure. The RNA extraction, PCR setup, PCR, and the post-PCR
manipulation (if required) are performed in separate laboratories. To reduce the
possibility of RNA degradation, the samples are processed into Trizol solution as
soon after collection as possible and usually within 24 h. All samples are stored
at room temperature before the addition of Trizol. We have found that storage at