Myeloid Leukemia

268 Martini, Teofili, and Larocca
3.2.1. Extraction of Granulocyte Total RNA
1. Incubate the samples (stored in Trizol) for 5 min at room temperature.
2. Add 0.2 mL of chloroform, shake vigorously for 3 min, and centrifuge at 11,000g
for 15 min at 4°C.
3. After centrifugation, the sample forms three phases: a red-colored lower phase, a
white interphase, and a colorless upper phase that contains total RNA. Transfer
approx 75–80% of the upper phase into a new tube and add 0.5 mL of isopropyl
alcohol.
4. Store the sample for 2 h at –20°C to facilitate RNA precipitation, then centrifuge
at 11,000g for 10 min at 4°C.
5. Discard the supernatant
6. Wash the RNA pellet with 1 mL of 75% ethanol (in DEPC-treated water), mix by
vortexing, and then centrifuge at 11,000g for 15 min at 4°C.
7. Remove the 75% ethanol and dry the RNA pellet at RT.
8. Resuspend the pellet in 30 µL of RNase-free water.
9. To quantify and to control for the quality of total RNA, each sample is subjected
to spectrophotometric analysis.
3.2.2. DNase Treatment
1. Add 1 µg of RNA to 1 µL of amplification-grade DNase I (Invitrogen), 1 µL of
10X DNase I reaction buffer, and RNase-free water up to a final volume of 10 µL.
2. Incubate the mixture for 15 min at room temperature, then stop the reaction by
adding 1 µL of 25 mM of EDTA (pH 8) and by heating the sample for 10 min at
65°C.
3.2.3. Phenol/Chloroform Purification and RNA Spectrophotometric
Measurement
1. After DNase treatment, dilute total RNA (10 µL) with 90 µL of RNase-free water
and add 100 µL of phenol/chloroform/isoamyl alcohol (25:24:1).
2. Vortex vigorously to obtain a lactescent mixture.
3. Centrifuge at 11,000g at 4°C for 10 min. After centrifugation, the mixture forms
two phases. Carefully transfer the upper phase, which contains the RNA, into a
new tube with 0.5 vol of ammonium acetate 7.5 M and 2 vol of 100% ethanol.
4. Incubate the sample for 2 h at –20°C, then centrifuge at 11,000g at 4°C for
10 min. The RNA forms a transparent pellet at the bottom of the tube.
5. Discard the supernatant, wash the pellet with 500 µL of 75% ethanol (in DEPCtreated
water), and centrifuge at 11,000g for 5 min at 4°C.
6. Remove the supernatant, dry the RNA at room temperature, then resuspend the
RNA in 8 µL of RNase-free water. To quantify and to control for the quality of
total RNA, each sample is subjected to spectrophotometric analysis.