Isolated PBMCs are cultured at 5 × 106 to 10 × 106 cells/ml for BLV quantification and 5 × 105 cells/ml for cell proliferation. Monocyte/macrophages are  isolated from PBMCs by exploiting their ability to adhere to plastic . After PBMCs are incubated for 4 to 6 h, nonadherent cells were removed and transferred to other wells. Isolated monocyte/macrophages are to be cultured with nonadherent cells separated by a transwell filter (0.4 μm pore size; Costar, Acton, Mass.). The purity of separated monocyte/macrophages is confirmed by flow cytometry with mouse anti-bovine CD14 immunoglobulin G (IgG; CAM36A; VMRD, Pullman, Wash.) and fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, Pa.) by using an EPICS profile analyzer. Adherent cells are  also stained for esterase as previously performed.  Cytoplasmic lysates from freshly isolated PBMCs are obtained by adding 200 μl of chloroform, and total RNAis  prepared by using TRI reagent according to the manufacturer’s protocol (MRC, Cincinnati, Ohio) with DNase treatment. The concentration of purified total RNA is  determined by spectrophotometry. Then, the RT reaction is  performed with purified total RNA. 

thanks Ravi its helpful

Did you try RNeasy spin columns?