Myeloid Leukemia

RNA and DNA From Leukocytes 11
3. Industrially produced plastics can be considered RNase-free, as long as they have
been handled with gloves. In addition, several buffers and solutions can be purchased
RNase-free.
4. Autoclaving does not inhibit, or only partially inhibits, RNase activity.
5. If glassware is used for the preparation and storage of buffers, inactivate RNases
by baking at over 250°C for several hours.
6. Solutions can be made RNase free by treatment with DEPC (caution, this compound
is toxic) (see Subheading 3.1.1.).
7. Because DEPC is highly unstable in the presence of Tris, buffers containing Tris
cannot be treated with DEPC. Use RNase-free components and DEPC-treated
water to prepare RNase-free buffers containing Tris.
8. After autoclaving, DEPC-treated buffers may smell sweet. This is caused by volatile
esters that are produced by the decomposing DEPC and is not a sign that the
DEPC has not been inactivated.
9. Repeated freezing and thawing of RNA or cDNA negatively affects its quality.
10. Because the target sequences that must be amplified may be located far away
from the 3� end of the gene, we recommend the use of random hexamers for the
cDNA reaction rather than oligo-dT primers.
References
1. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by
acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,
156–159.
2. Glisin, V., Crkvenjakov, R., and Byus, C. (1974) Ribonucleic acid isolated by
cesium chloride centrifugation. Biochemistry 13, 2633–2637.
3. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979)
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry 18, 5294–5299.
4. Miller, S. A., Dykes, D. D., and Polesky, H. F. (1988) A simple salting out procedure
for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215.