Myeloid Leukemia

80 Branford and Hughes
cell lines together, only two controls are required to monitor both the b2a2 and
b3a2 assays.
1. Using standard tissue-culture techniques, the cells are grown until there are enough
cells to provide an adequate supply of control material to last several years.
2. The low control is prepared by diluting the cells at a ratio of: 5 × 10 2 K562 cells
: 5 × 10 2 Molm-1 cells : 5 × 10 6 Hela cells.
3. The high control is prepared by diluting the cells at a ratio of: 4 × 10 5 K562 cells
: 6 × 10 5 Molm-1 cells : 4 × 10 6 Hela cells.
4. The diluted cell mixture is centrifuged at 2000g to pellet the cells, which are then
washed twice in phosphate-buffered saline.
5. Dissolve the pellet in an adequate volume of Trizol solution to give approx 5 ×
10 6 cells in 1.6 mL. Draw the solution through an 18-gauge blunt needle repeatedly
to shear the DNA.
6. Add 1.6 mL of the solution to 2-mL tubes and store at –80°C.
7. The Hela cells are also used as a negative control. Aliquots of 2 × 10 6 Hela cells
are stored at –80°C in 1.6 mL of Trizol.
8. As required, a tube of each control is removed from the freezer and the RNA is
extracted as outlined under Subheading 3.1., and cDNA is prepared as outlined
under Subheading 3.2.
3.5. Real-Time Quantitative RT-PCR
Three separate RQ-PCR reactions are prepared for the b2a2 BCR-ABL transcript,
the b3a2 BCR-ABL transcript, and the BCR control gene. The results are
reported as a ratio of BCR-ABL/BCR%, which compensates for variations in
the quality of the RNA sample and for differences in the efficiency of the RT
(see Note 2). The probes and primers were selected using the Primer Express
software following the manufacturer’s guidelines (see Note 3). The probes are
labeled with a 5� FAM reporter dye and a 3� TAMRA quencher dye. The three
assays are processed in the same 96-well reaction plate, because the same PCR
reaction conditions are used for each. Each patient cDNA duplicate sample is
processed once within two separate quantitative runs. Therefore, the samples
are processed in duplicate and the results of the two RQ-PCR analyses are
averaged. The standards, controls, and no-template control are processed once
within each run, except for the lowest standards for the b2a2 and b3a2 BCR-
ABL transcripts (representing approx 10 copies per 2.5 µL). These standards
are run in duplicate due to the wider variability of the assay at low copy number
(see Note 4). The preparation of the PCR reactions and the addition of the
cDNA are performed in separate laboratories. An Excel spreadsheet is prepared
for each assay, and the cells within the spreadsheet correspond to the 96well
reaction plate. The location of each standard, patient sample, and control
within the plate is marked, and the spreadsheet is printed and used during PCR
preparation to guide the loading of the wells. The TaqMan analysis uses fluo-