Myeloid Leukemia

RNA and DNA From Leukocytes 5
late RNA from mammalian cells exist. A commonly used protocol, on which
various commercially available reagents (like Trizol ® or RNABee ® ) are based,
involves the use of acid guanidinium thiocyanate–phenol–chloroform extraction
(1). A second procedure involves the lysis of cells in denaturing
guadinidium thiocyanate solution and separation of RNA by ultracentrifugation
on a 5.7 M CsCl cushion (2,3). The first method is very well suited for the
simultaneous processing of multiple samples, and may be preferable in a routine
diagnostic setting. The latter method yields very high-quality RNA, and
allows the isolation of RNA from large numbers of cells in one tube. The drawbacks
of this method are the long centrifugation times and the limited number
of samples that can be processed in one run (most ultracentrifuge rotors fit six
tubes). In addition, if open ultracentrifuge tubes are used (rather than the more
difficult to handle sealable tubes), there may be an increased risk of crosscontamination.
When handling RNA, it is important to take precautions to
avoid degradation by ribonuclease activity (see Notes 2–8).
3.1.1. Decontamination of Solutions From Ribonuclease (RNAse) Activity
Using Diethylpyrocarbonate (DEPC)
RNase activity can be inactivated by treatment of solutions with DEPC. Note
that DEPC is toxic, and that buffers containing Tris should not be treated with
DEPC (see Notes 6–8).
1. Add 0.1% DEPC (w/v).
2. Mix thoroughly and leave overnight at 37°C.
3. Because DEPC may also inhibit subsequent enzyme activity necessary for cDNA
and PCR, it should be removed by autoclaving for 30 min, which causes hydrolysis
of the DEPC (see Notes 6–8).
3.1.2. RNA Isolation Using Commercially Available Complete Reagent
Under this subheading, the isolation of total RNA using RNABee reagent is
described. Several other commercially available kits are on the market that
may be used as well.
1. If working with freshly isolated nucleated cells, proceed to step 2. If
cryopreserved cells are used, rapidly thaw the cells at 37°C. Transfer them to a
15-mL tube on ice. Add medium (e.g., phosphate-buffered saline [PBS], HBBS,
Dulbecco’s modified Eagle’s medium [DMEM]) supplemented with 1% fetal calf
serum (FCS; 4°C) up to a total volume of 15 mL.
2. Spin down the cells at 350g for 5 min at 4°C. Discard the supernatant and resuspend
the pellet in the remaining solution.
3. Add 10 mL ice-cold medium (PBS, HBBS, DMEM) with 1% FCS, and count the cells.
4. Spin down cells at 350g for 5 min at 4°C. Discard the supernatant and resuspend
the pellet in the remaining solution.