Myeloid Leukemia

82 Branford and Hughes
a. 1 cycle at 50°C for 2 min.
b. 1 cycle at 95°C for 10 min.
c. 40 cycles at 95°C for 15 s, 60°C for 1 min.
13. The PCR is completed in approx 2 h. Following completion, save the run. Check
each well of the reaction plate to ensure that evaporation has not occurred and
then discard the plate.
14. The SDS software is used to analyze the PCR results. The standard curves are
calculated and the patient and control results are printed on an experiment report
(see Note 4).
15. Results are reported as a percentage ratio of BCR-ABL/BCR%. The quality control
values are accepted according to Westgard Quality Control rules (28) (see
Note 5).
16. The level of BCR transcript determines the quality of RNA. Values greater than
30,000 transcripts are adequate and produce appropriate BCR-ABL/BCR% results.
3.6. Qualitative PCR for Atypical BCR-ABL Transcripts
For patients with CML where the usual p210 transcripts are not detected, we
perform a qualitative PCR that amplifies a region spanning BCR exon 1 and
ABL exon 3 in order to identify atypical BCR-ABL transcripts. As quality control
material, the RNA of patients with the p210 and p190 BCR-ABL transcripts
are used. The preparation of the PCR reactions and the addition of the cDNA
are performed in separate laboratories.
1. The primers used in the PCR are:
Forward Primer: 5� acc gca tgt tcc ggg aca aaa g
Reverse Primer: 5� tgt tga ctg gcg tga tgt agt tgc ttg g (29).
2. The patient RNA is extracted as outlined under Subheading 3.1., and cDNA is
prepared as outlined under Subheading 3.2.
3. The quality of the cDNA is confirmed by quantitation of the BCR control gene by
RQ-PCR analysis as outlined under Subheading 3.5. Samples with BCR values
less than 30,000 transcripts are not of sufficient quality for analysis.
4. Thaw the PCR reagents and primers and mix thoroughly before use (except for
the enzyme, which is stored at –20°C until added to the master mix). Centrifuge
briefly to collect the material at the bottom of the tube.
5. Prepare a master mix on ice by adding the following into a 1.5-mL tube for each
patient sample + 2 positive controls + 1 no-template control + 1: 1.5 µL MgCl 2,
2.5 µL dNTP (10 mM mix); 5 µL 10X Expand long template Buffer 3; the forward
and reverse primers at a final concentration of 0.3 µM, 0.75 µL of 5 U/µL
Expand long template enzyme mix and sterile water to a total volume of 47 µL.
6. Mix the tubes thoroughly and centrifuge briefly to collect the mix at the bottom
of the tube.
7. Pipet 47 µL of the master mix into 0.2-mL tubes.
8. In a separate laboratory, add 3 µL of the cDNA, mix thoroughly, and centrifuge
briefly to collect the mix at the bottom of the tube.