Nuclei Isolation from Bone Cells for Nuclear Run-on Assays

Nuclear run-on assay is the most sensitive method to measure specific gene transcription. This assay is used to determine whether a change in the mRNA level of a specific gene results from a change in its synthesis. The nuclear run-on assay consists of four steps: (i) isolation of intact nuclei, (ii) incubation of nuclei with [32P]UTP and unlabeled ATP, CTP and GTP to label the nascent RNA, (iii) purification of the labeled RNA and (iv) detection of specific mRNA by hybridizing the labeled RNA with the corresponding genomic DNA or cDNA immobilized onto a nitrocellulose membrane. The important step in the nuclear run-on assay is obtaining intact nuclei free of cell membrane and cytoplasmic materials, because the presence of these nonnuclear constituents results in poor incorporation of [32P]UTP into nascent RNA. Several methods are used to isolate intact nuclei, and the preferred method of isolation depends on the cell type. Most methods involve swelling the cells in a hypotonic buffer followed by detergent lysis (usually Nonidet P40 [NP40]), Dounce homogenization or both, with or without pelletting through 2 M sucrose by ultracentrifugation to free nuclei from cell membrane and cytoplasmic materials (3). Nuclei from fragile cell lines and primary cultures like lymphocytes and thymocytes are isolated by cell lysis in iso-osmotic solution with or without NP40, followed by ultracentrifugation through a 2 M sucrose cushion (2–4). In our studies of rat clonal osteoblast-like cells (ROS 17/2.8 and UMR 106-01), we found that none of the published methods produced intact nuclei from these cells. Moreover, to our knowledge no published reports exist describing nuclear run-on analysis of clonal cells derived from bone. We describe a simple procedure for isolating intact nuclei from these cells for a nuclear run-on assay. This procedure is a modification of the hypotonic/NP40 lysis procedure (3). The following procedure is for 1 × 106 to 5 × 107 cells. Wash the monolayers twice with 5 mL of ice-cold phosphate-buffered saline, scrape and centrifuge in a 50-mL polypropylene tube at 500× g for 5 min at 4°C. Resuspend the cell pellet in 40 mL of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 3 mM CaCl2 and 3 mM MgCl2), and centrifuge at 500× g for 5 min at 4°C. Resuspend the cells by vortex mixing the cell pellet gently for 5–10 s (vortex speed at 3 or 4) while adding 1 mL of lysis buffer dropwise, and leave the tube in ice for 10 min. Transfer the cell suspension to an ice-cold Dounce homogenizer, add 1 mL of NP40 lysis buffer (1% NP40 in lysis buffer), and break the cells with 20–25 strokes of a tight-fitting pestle. Overlay the homogenate on 6 mL of a 1 M sucrose cushion (1 M sucrose in lysis buffer) in a 14-mL polypropylene tube (17 × 100 mm, Falcon; Becton Dickinson Labware, Bedford, MA, USA), and centrifuge at 500× g for 30 min (either a fixed-angle rotor or swinging-bucket rotor can be used). Remove the supernatant carefully using a Pasteur pipet with suction, and suspend the pellet in 0.2 mL of ice-cold glycerol storage buffer (50 mM Tris-HCl, pH 8.3, 5 mM MgCl2, 0.1 mM EDTA and 40% glycerol) by pipetting up and down 6–10 times with a 1-mL micropipet at 0.2 mL. Cover the tube with its cap, and store at -70°C or in liquid nitrogen. The nuclei appear intact when seen under a microscope, and the recovery is about 75% as determined by hemacytometer. Figure 1A shows the result of a nuclear run-on assay using nuclei isolated from ROS 17/2.8 cells treated with transforming growth factor β (1 ng/mL) for 48 h. Labeling of nascent RNA transcripts and purification of RNA were carried out as described (3) with the following modifications. Frozen nuclei (200 μL) stored in a 14-mL polypropylene tube were thawed at room temperature, 200 μL of 2× reaction buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 300 mM KCl, 5 mM dithiothreitol, 1 mM each of ATP, CTP and GTP) and 200 μCi of [α-32P]UTP (800 Ci/mmol, 10 mCi/mL) were added, and the reaction mixture was incubated at room temperature for 30 min with occasional