Myeloid Leukemia

144 Mokany et al.
or taken out of the rooms). Primer and reagent (Tables 1 and 2) handling should
be made in a PCR setup room. Calibrator and RNA manipulations (see Subheading
3.1.) should be made in a designated template room (DNA and RNA extractions
also take place here), and thermocyclers (see Subheading 3.3.) should be
kept in a separate room. Extraction or manipulation of concentrated plasmid stock
(above the concentration of the top calibrator) or PCR product should be done in
an area removed from all others, and preferably in a designated room. Meticulous
laboratory protocol should be followed to avoid contamination. Clean benches
with 0.05% hypochlorite and 70% ethanol before starting. Clean all pipets with
70% ethanol. Change gloves frequently. Centrifuge all tubes before use to prevent
production of aerosols. Minimize sample handling. In the designated PCR
rooms, we use disposable laboratory coats, hair nets, shoe covers, and gloves at
all times, to ensure no possible carryover between rooms and the lab.
8. As a result of variation in expected abundance of the PML-RARα transcript in
diagnostic (pretreatment) compared with follow-up (posttreatment) patient
samples, the amount of patient RNA amplified per reaction varies accordingly. A
total of 50 ng RNA is analyzed for pretreatment specimens, and 500 ng for specimens
collected following treatment.
9. All steps using the QIAamp RNA Blood Mini Kit are carried out at room temperature.
Working quickly will minimize degradation of RNA.
10. If the volume required for M-MLV is too small to accurately pipet, the M-MLV
may be diluted 10-fold in 1X PCR buffer.
11. The QZyme method described in this chapter was individually developed. As a
result, the details of the bulk mix and thermocycling conditions are non-standard.
Please follow the instructions as described in this chapter and disregard buffers and
thermocycling programs recommended in published BD Bioscience protocols.
12. Use a new pipet tip for each addition of master mix and RNA calibrators to the wells.
Addition of the master mix can be done with a multi-displacement pipet such as the
Eppendorf Research ® pro. All reactions should be performed at least in duplicate.
13. To ensure adequate activity of the reverse transcriptase, polymerase, and other
reaction components, control reactions should be included in every RT-PCR assay.
Appropriate controls include (1) positive controls for the target (calibrators
for each target), (2) no amplification controls (NAC) to ensure assay specificity
(RNA from a cell line devoid of the target), and (3) no-template controls (NTC)
to check for cross-contamination (water instead of target). The reverse transcription
of the PML-RARα transcript is indirectly controlled for by the RT-PCR of
the BCR transcript in the same tube. The authors acknowledge that the activity of
the reverse transcriptase on the BCR transcript from calibrators is indirect evidence
for its activity on the PML-RARα transcript and is therefore not ideal.
14. Correct procedure for setting up the ABI 7700 for a duplex reaction can be found
at the following link:
www.appliedbiosystems.com/support/tutorials/pdf/setting_up_duplex_reactions.pdf
Collection of fluorescent data at the annealing step only can be performed to
reduce the size of run files.