Myeloid Leukemia

PRV-1 Overexpression in PV and ET 267
24. 10X reaction buffer A without MgCl 2: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl,
0.1 mM EDTA, 1 mM DTT, 50% glycerol, and 1% Triton X-100. (Promega).
25. Oligonucleotide primers (10 pM) (Invitrogen).
26. Agarose and electrophoresis equipment.
27. 10X TBE buffer: 890 mM Tris, 890 mM boric acid, 0.01 M EDTA (pH 8.3).
28. Ethidium bromide (10 mg/mL).
3. Methods
The following methods outline (1) the purification of peripheral blood granulocytes,
(2) RNA extraction and DNase treatment, (3) reverse transcription and testing
to verify the presence of cDNA, and (4) PRV-1 amplification and detection.
3.1. Purification of Peripheral Blood Granulocytes
Purification of peripheral blood granulocytes is described under Subheadings
3.1.1. and 3.1.2. This includes (a) the collection and storage of peripheral
blood and (b) the purification of granulocytes.
3.1.1. Collection and Storage of Peripheral Blood
Fifteen milliliters of peripheral blood are collected in sodium citrate and stored
at 4°C, until they can be diluted with an equal amount of Ca 2+ - and Mg 2+ -free PBS.
3.1.2. Purification of Peripheral Blood Granulocytes
1. In order to remove red cells, mix 1 mL of Hespan with 7 mL of cell suspension
and then sediment for 90 min at room temperature.
2. Harvest the upper layer containing all the nucleated cells, transfer it into a new
tube, and centrifuge at 1200g for 10 min at 4°C.
3. Wash the pellet twice with 10 mL of PBS and resuspend the pellet in 9 mL of
PBS. Then carefully layer the cell suspension over 5 mL Ficoll-Hypaque density
gradient, and centrifuge at 1800g for 20 min.
4. Recover all the cells at the bottom of the vial. This population should consist of
over 95% granulocytes (see Note 1). Wash these cells twice in 10 mL of PBS
with centrifugation at 1200g for 10 min.
5. Resuspend the pellet of highly purified granulocytes in 1 mL of Trizol reagent.
Samples suspended in Trizol can be stored prior to PRV-1 analysis for several
months at –80°C, or for 2 mo at –20°C.
3.2. RNA Extraction of Highly Purified Granulocytes
and DNase Treatment
RNA extraction and DNase treatment is delineated under Subheadings
3.2.1.–3.2.3. This includes (a) the extraction of granulocyte total RNA, and its
measurement by spectrophotometry, (b) DNase treatment, (c) phenol/chloroform
purification, and a second spectrophotometric measurement of RNA.