Myeloid Leukemia

180 Cools, Stover, and Gilliland
cell lysis buffer and repeat the procedure from step 2. Keep the cells on ice all the
time.
5. Aspirate the supernatant and resuspend the white blood cells in cold PBS.
6. Remove an aliquot to count the cells.
7. Pellet 5 × 10 6 to 10 7 cells by centrifugation (450g for 10 min at 4°C) (see Note 3).
8. Keep the cells on ice. Remove the supernatant, and vortex the cell pellet for 5 s.
Proceed with RNA/DNA or protein extraction.
3.1.2. Simultaneous RNA and DNA Extraction From White Blood Cells
1. Add 1 mL Trizol (Invitrogen) to the cells and pipet up and down several times
(see Note 4).
2. Incubate the lysate at room temperature for 5 min.
3. Add 200 µL of chloroform.
4. Shake the mixture vigorously for 15 s.
5. Incubate at room temperature for 10 min.
6. Centrifuge at 12,000g for 15 min at 4°C.
7. After centrifugation, the mixture is separated in a colorless upper phase, a white
interphase, and a red lower phase. The RNA is present in the aqueous upper
phase. Transfer the colorless upper phase (600 to 650 µL) to a clean 1.5-mL tube.
Avoid touching the interphase in order not to risk contamination of the RNA with
DNA. Save the interphase and the lower phase for DNA extraction.
8. Add 500 µL of isopropanol to the colorless phase. Invert the tube three times and
incubate at room temperature for 10 min. While incubating the RNA/isopropanol
mixture, use the following method for DNA extraction:
a. Add 300 µL of 100% ethanol to the interphase and red lower phase (these
phases contain the genomic DNA and proteins). Invert the tube three times. A
precipitate should form directly. Incubate 3 min at room temperature.
b. Centrifuge at 2000g for 5 min at 4°C.
c. Discard the supernatant, and wash the pellet three times with DNA wash solution.
Incubate for 20 min in this solution. Invert the tube several times during
this wash step.
d. Collect the DNA pellet by centrifugation at 2000g for 5 min at 4°C.
e. Repeat steps c and d two more times.
f. After these three wash steps, perform a final wash step with 70% ethanol.
g. Remove all ethanol and air-dry the DNA pellet for 5 min.
h. Resuspend the DNA in 8 mM NaOH. Do not pipet up and down too much,
because this will break the DNA.
i. Let the DNA dissolve at 4°C overnight.
j. Add HEPES to adjust the pH (see Note 5), and measure the concentration and
A 260/A 280 ratio.
9. Centrifuge at 12,000g for 20 min at 4°C.
10. After centrifugation, a small, white RNA pellet is visible. Carefully discard the
supernatant and add 1 mL 70% ethanol (make sure RNase-free water is used to
dilute the ethanol). Invert the tube five times.