Myeloid Leukemia

160 Tobal and Yin
4. Notes
1. Some samples may contain cellular clumps; if this is the case, syringing the cells/
solution D mix (after Subheading 3.1., step 2) with an approx 21-gauge needle
could help to disperse the cells and aid cellular lysis.
2. Genomic DNA contamination may be precipitated with the RNA. High levels of
genomic DNA contamination could therefore affect RT-PCR results. A second
round of extraction (repeat the protocol previously described) could help in eliminating
or significantly reducing the level of genomic DNA contamination.
3. It is necessary to perform all preparation in a sterile cabinet to avoid cross-contamination.
4. M-MLV reverse transcriptase is a thermolabile enzyme and should be kept at all
times on ice while in use, then stored at –20°C.
5. The performance of positive and negative control RT reactions is necessary for
accurate analysis.
6. Although a shorter RT reaction could be performed, this tends to produce lower
levels of cDNA.
7. It is necessary to perform all preparation in a sterile cabinet to avoid cross-contamination.
8. Negative RT-PCR results for the control gene (ABL) indicate a lack of goodquality
amplifiable RNA or cDNA. If this is the case, re-extraction of the RNA
and the performance of a new RT reaction may help.
9. Positive RT-PCR results in negative control reactions indicate cross-contamination,
and the analysis should be repeated.
10. It is necessary to perform all preparation in a sterile cabinet to avoid cross-contamination.
11. Once the cDNA is prepared, the volume is then diluted to 50 µL with water.
Using 5 µL of cDNA in the RQ-PCR reaction reduces variation in the level quantified
due to sample handling.
12. Performing a standard curve quantification is necessary with all new sets of primers
and probes.
13. A low level of ABL indicates a low amount of amplifiable RNA/cDNA.
Re-extraction of RNA may help in improving the results.
References
1. Raimondi, S. C., Kalwinsky, D. K., Hayashi, Y., Behm, F. G., Mirro, J. Jr., and
Williams, D. L. (1989) Cytogenetics of childhood acute nonlymphocytic leukemia.
Cancer Genet. Cytogenet. 40, 13–27.
2. Rowley, J. D. (1990) Recurring chromosome abnormalities in leukemia and lymphoma.
Semin. Hematol. 27, 122–136.
3. Erickson, P., Gao, J., Chang, K. S., et al. (1992) Identification of breakpoints in
t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/
ETO, with similarity to Drosophila segmentation gene, runt. Blood 80, 1825–
1831.