Concurrent isolation of ribosomal, messenger, and low molecular weight RNA.

The analysis of the cellular and molecular metabolism of anticancer drugs has provided important information for optimizing the design of clinical trials (9). In recent years, there has been an increasing use and development of RNAdirected chemotherapeutic agents, such as ribonucleotide analogs (3,5,11,13), actinomycin D (14), and flavopiridol (7). To understand their mechanisms of action, it is essential to analyze each of the various RNA species because the compound may be differentially incorporated and/or exert a dissimilar activity on the three RNA polymerases (8). Such an analysis can be achieved through the examination of the three main classes of RNA (rRNA, mRNA, and low molecular weight RNA, sRNA, which includes 5S and tRNA). Historically, this type of analysis was performed either by using an in vitro system in which the three RNA polymerases were isolated and assayed separately (8) or by isolating and crudely fractionating total RNA through a sucrose gradient (15). There are numerous techniques and kits available for the isolation and separation of mRNA (1,6,10), but they do not allow for the concurrent separation of rRNA and sRNA. The three RNA species can be separated and isolated using high-pressure liquid chromatography (2), but the costs of the instruments and columns are beyond the reach of most researchers. The in vivo and ex vivo effects of RNA-directed compounds on each of the three RNA species can be characterized by using a simultaneous separation method. Here we describe a method for RNA isolation that allows for the simultaneous separation and enrichment of mRNA, rRNA, and sRNA. This procedure utilizes standard oligo(dT) chromatography for mRNA isolation while the sRNA and rRNA are isolated by subjecting the non-polyadenylated RNA to anion-exchange chromatography. This was accomplished by combining and modifying three strategies for isolating various RNA forms (Figure 1). Specifically, 1 × 108 cells from the multiple myeloma cell line, MM.1S (4), were pelleted by centrifugation. The pellets were either used directly or flash-frozen on dry ice and stored at -80°C. The fresh or flashfrozen cell pellets were lysed in 15 mL FastTrack 2.0 lysis buffer (200 mM NaCl, 200 mM Tris, pH 7.5, 1.5 mM MgCl2, 2% SDS; proprietary mixture of proteases) and digested per the manufacturer’s instructions (Invitrogen, Carsbad, CA, USA). This cell lysate was used to separate the three RNA species (Figure 1, no. 1). The lysate was first applied to 75 mg oligo(dT) cellulose (FastTrack 2.0) to separate and enrich the mRNA per the manufacturer’s protocol, with the following modifications (Figure 1, no. 2). The oligo(dT) cellulose was pelleted at room temperature by centrifugation for 5 min at 5000× g. The supernatant was removed and saved on ice for further isolation of rRNA and sRNA. To wash the mRNA-bound cellulose, the pellet was resuspended in 20 mL Binding Buffer (500 mM NaCl, 10 mM Tris-Cl, pH 7.5) and centrifuged as before. The 20-mL wash supernatant was combined and saved with the 15-mL binding supernatant. The pellet was again washed with 10 mL Binding Buffer, but the supernatant from this wash was discarded. To remove nonspecifically bound RNA, the pellets were washed approximately five times in 10 mL Low Salt Wash Buffer (250 mM NaCl, 10 mM Tris-Cl, pH 7.5) and centrifuged as before. The 10-mL supernatant from the first Low Salt Wash was combined and saved with the previously saved supernatant. The remainder of the mRNA isolation was performed according to the manufacturer’s protocol (Figure 1, no. 3). The mRNA (10–30 μg) was resuspended in 20 μL DEPC-treated water. The supernatants that had been saved from the mRNA isolation, approximately 45 mL, were precipitated with equal volume of isopropanol at -20°C overnight and centrifuged at 12 000× g for 30 min at 4°C in a Sorval ss-34 rotor. The supernatant was discarded, and the pellets were washed with 70% ethanol (Figure 1, no. 4). This pelleted material contained rRNA, sRNA, genomic DNA, proteins, and other cellular materials. The pellet was allowed to air dry briefly, and the RNA was isolated from this mixture by extraction with 10 mL RNAzol B (Teltest, Friendswood, TX, USA) per the manufacturer’s instructions, with all centrifugations performed at 12 000× g for 15 min at 4°C in a Sorval ss-34 rotor (Figure 1, no. 5). This extraction allows for the removal of the DNA, protein, and other cellular contaminates. In addition, incorporating this step permits the quantitation of the RNA before applying it to the anion exchange resin. This helps to maximize the amount of DRUG DISCOVERY AND GENOMIC TECHNOLOGIES