Myeloid Leukemia

Quantitative PCR for Monitoring CML Patients 75
4. Expand Long Template PCR System (Roche). Store at –20°C.
5. Agarose, molecular-biology grade (Progen). Store at room temperature.
6. SPP1/EcoR1 DNA molecular-weight marker (Geneworks). Store at –20°C.
2.7. Qualitative PCR for p210 BCR-ABL
1. Primer Express software (Applied Biosystems).
2. PCR primers. All primers are ordered as a 50 µM solution and stored in 30-µL
lots at –20°C.
3. 10 mM stock of dNTPs (Amersham Biosciences). Store at –20°C in 100-µL lots.
4. 25 mM MgCl 2 (Applied Biosystems). Store at –20°C.
5. 10X PCR buffer (Applied Biosystems). Store at –20°C.
6. AmpliTaq Gold (Applied Biosystems). Store at –20°C.
7. Agarose, molecular-biology grade (Progen). Store at room temperature.
8. Puc19/Hpa11 DNA molecular-weight marker (Geneworks). Store at –20°C.
3. Methods
3.1. RNA Extraction
1. Isolate the white cells from 10 to 20 mL of peripheral blood. Pour the whole
blood into a 50-mL tube. Aliquot 70 mL of lysis buffer into a clean UV-irradiated
beaker (use a clean beaker for every patient). Pour lysis buffer into the 50-mL
tube from the beaker to a volume of 50 mL and mix or stand for 10 min. Centrifuge
for 10 min at 2000g to pellet the white cells.
2. Carefully remove the supernatant by using a waste vacuum system and a sterile
mixing cannula, without disturbing the white cell pellet. Remove as much of the
lysed cells as possible without disturbing the white cell pellet. If processing more
than one patient sample, rinse the vacuum waste system inlet with water and
replace the cannula for each sample. Add 20 mL lysis buffer and resuspend the
white cells with a sterile transfer pipet. Mix or stand for 5 min and repeat the
centrifugation. Remove the supernatant, leaving the white cell pellet.
3. As a general rule for white cells derived from 20 mL of blood, 3.2 mL of Trizol
reagent is sufficient to stabilize the RNA. For patients at diagnosis or at relapse,
large white cell pellets may require either reduction of the pellet or addition of
extra Trizol. The extra Trizol reagent must be sufficient to completely dissolve
the white cells. We have successfully adopted these guidelines to determine the
amount of Trizol rather than basing the volume on the white cell concentration,
which is not practical to measure in our laboratory. Add the appropriate amount
of Trizol to the 50-mL tube and draw the solution up and down six times into a
blunt 18-gauge needle attached to a 3-mL syringe (a blunt needle avoids the risk
of needle-stick injury). This will lyse the white cells and shear the DNA, which
reduces the risk of DNA contamination during the RNA extraction procedure. If
white cells are still present after mixing, add extra Trizol and mix until the white
cells are lysed. If the Trizol mixture is extremely viscous, add extra Trizol until
the viscosity is reduced.