Myeloid Leukemia

Quantitative PCR for Monitoring CML Patients 77
set-up laboratories. The reverse transcription procedure is performed in duplicate
for each patient RNA.
1. To a sterile 0.2-mL tube add 2 µL of random hexamers (25 µM final concentration)
and a volume of DEPC water to give a total volume of 2 µg of RNA in 9 µL
of DEPC water.
2. In the PCR cabinet, add 2 µg of RNA and mix by drawing repeatedly into the
pipet tip. Transfer the tubes to the thermal cycler and heat the mixture to 70°C for
10 min. Quick-chill on ice and return the tubes to the PCR cabinet.
3. Prepare a master mix of the SuperScript II reagent components. For each sample,
add 4 µL of 5X First Strand Buffer, 2 µL of 0.1 M DTT, and 1 µL dNTP mix
(10 mM each).
4. Add 7 µL of the master mix to each sample and mix by drawing into the pipet tip.
Briefly centrifuge the tubes at 12,000g to collect the reactants in the bottom of
the tube.
5. Transfer the tubes to the thermal cycler and incubate at 42°C for 2 min. Pause the
reaction and return the tubes to the PCR cabinet.
6. Remove the SuperScript II from the freezer. Mix and centrifuge briefly at 12,000g
to collect the solution at the bottom of the tube. Place the SuperScript II on ice.
7. Add 2 µL of SuperScript II to each tube in the PCR cabinet. Mix well by drawing
into the pipet tip. Briefly centrifuge the tubes at 12,000g and transfer the samples
into the thermal cycler.
8. Continue the reaction at 25°C for 10 min, 42°C for 50 min, 70°C for 15 min, and
hold at 4°C.
9. After completion of the reaction, the samples may be stored at –20°C until
required.
3.3. Plasmid Standards
Plasmids containing a cDNA fragment of the genes under analysis are prepared
by PCR cloning. The plasmids are used to construct standard curves for
quantitation of each of the target and control genes. In our quantitative PCR
assay, we prepared plasmids for the quantitation of the b2a2 BCR-ABL transcript
and the BCR control gene from a cell line expressing the b2a2 BCR-ABL
transcript (KCL22). A plasmid for the quantitation of the b3a2 BCR-ABL transcript
was prepared by N. Cross (27). This plasmid contains a modified b3a2
BCR-ABL transcript derived from the K562 cell line. Approx 200 bases are
removed from ABL exons 2 and 3, and 100 bases of a different sequence ligated
in that region. The plasmid contains an intact sequence in the region of the
sequence used for quantitation of the b3a2 transcript in our assay. However, a
standard for the b3a2 BCR-ABL transcript can be prepared if required by following
the same procedure as for the b2a2 BCR-ABL transcript and using a
b3a2 BCR-ABL expressing cell line.