Myeloid Leukemia

RNA and DNA From Leukocytes 7
9. Using an RNase-free disposable pipet, remove most of the supernatant, including
the interface.
10. Invert the tubes to remove the remaining fluid and cut off the round bottom of the
tube containing the pelleted RNA using an RNase-free razor blade.
11. Resuspend the RNA (which should be visible as a clear gelatinous pellet) in 200 µL
DEPC-treated H 2O, and transfer to an Eppendorf tube.
12. Rinse the bottom of the cut polyallomer tube twice with 100 µL H 2O, and transfer
to the Eppendorf tube.
13. To the 400 µL RNA solution add 40 µL 3 M sodium acetate (pH 5.2) and precipitate
the RNA by adding 1 mL ethanol. Store for at least 30 min at –80°C and
centrifuge for 10 min at 14,000g; discard the supernatant and dissolve the pellet
in 400 µL H 2O.
14. Repeat the precipitation (step 13) once and resuspend the pellet in 100 µL DEPCtreated
H 2O.
15. Determine the amount of RNA by measuring the OD 260 and OD 280. The concentration
of RNA (µg/mL) = OD 260 × dilution factor × 40. Ideally, the OD 260/OD 280
ratio should be 2.0.
16. As the OD gives only a rough indication of the quality of the sample, determine
the quality of the RNA on a 1.5% agarose gel (see Subheading 3.1.4.).
17. Store RNA samples at –20°C or –80°C in water. If preferred, RNA can also be
stored as an ethanol precipitate (add 0.1 volume of 3 M sodium acetate, pH 5.2,
and add ethanol to a final concentration of 70%). Note that repeated freeze-thawing
will negatively affect the quality of the RNA (see Note 9).
3.1.4. Quality Control of RNA by Gel Electrophoresis
The quality of RNA can be judged by visualization of 18S and 28S ribosomal
RNA bands after gel electrophoresis. Instead of using formaldehyde-containing
gels that are normally used for Northern blotting, a simple and fast
protocol is described below using regular 0.5X TBE-containing agarose gels
and SDS-containing loading buffer.
1. Pour a fresh 1.5% agarose gel in 0.5X TBE buffer containing 3.5 µL ethidium
bromide per 100 mL.
2. Dilute 0.5–1 µg RNA in 10 µL H 2O.
3. Add 2 µL 6X loading buffer (dissolve 0.25 g bromphenol blue, 0.25 g xylene
cyanol, and 15 g Ficoll in 100 mL H 2O, and add 10% SDS w/v).
4. Load the RNA on the gel and electrophorese in 0.5X TBE buffer.
5. Visualize the RNA using UV illumination. If the RNA is intact, the 18S and 28S
ribosomal RNA should appear as two discrete bands, with the 28S band being
approximately twice as intense as the 18S (Fig. 1).
3.2. Isolation of DNA
DNA can be extracted using many different protocols. For Southern blotting,
high-molecular-weight DNA should be isolated, which can be achieved