Myeloid Leukemia

Quantitative PCR for Monitoring CML Patients 81
rescent probes; therefore, powder-free gloves must be used during all steps of
the procedure and the tubes or plates must not be marked with a fluorescent
marking pen.
1. The sequence of the primers and probes are:
b2a2 BCR-ABL:
Forward primer: 5� atc cgt gga gct gca gat g
Reverse primer: 5� cgc tga agg gct tct tcc tt
TaqMan probe: 5� cca act cgt gtg tga aac tcc aga ctg tcc
Amplicon length 96 bp
b3a2 BCR-ABL:
Forward primer: 5� ggg ctc tat ggg ttt ctg aat g
Reverse primer: 5� cgc tga agg gct ttt gaa ct
TaqMan probe: 5� cat cgt cca ctc agc cac tgg att taa gc
Amplicon length 74 bp
BCR:
Forward primer: 5� cct tcg acg tca ata aca agg at
Reverse primer: 5� cct gcg atg gcg ttc ac
TaqMan probe: 5� tcc atc tcg ctc atc atc acc gac a
Amplicon length 67 bp
2. The probes and primers are thawed and mixed thoroughly before use. Centrifuge
briefly to collect the material at the bottom of the tube.
3. Prepare master mixes for each transcript by adding the following to a 1.5-mL
tube for each patient sample + 2 positive controls + 2 negative controls + 1: 12.5 µL
2X TaqMan universal master mix, the forward and reverse primers at a final concentration
of 0.2 µM, the probe at a final concentration of 0.1 µM and sterile water
to a total volume of 22.5 µL. A no-template control is included for each mix.
4. Mix the tubes thoroughly and centrifuge briefly to collect the mix at the bottom
of the tube.
5. Pipet 22.5 µL of each master mix into the appropriate wells of the 96-well plate.
6. In a separate laboratory, thaw the standards. Mix and centrifuge briefly to collect
the standard at the bottom of the tube.
7. In the PCR cabinet, add 2.5 µL of each standard, patient sample, and control into
the appropriate wells.
8. Cap the wells with strips of optical caps.
9. Centrifuge the 96-well plate briefly to collect the mix at the bottom of the wells.
10. Load the PCR plate into the 7700 Sequence Detector System. Refer to the
manufacturer’s user manual for instructions for use of the instrument.
11. A template is prepared on the analyzer for the set-up of the RQ-PCR that has the
positions and concentrations of the standards already indicated. The reaction volume
and thermal cycler conditions are preprogrammed. The template is copied
for each new run, and the position and identification of the patient samples, controls,
and no-template controls are entered. Once the template copy is programmed,
the run is started.
12. The thermal cycling conditions for each reaction are: