Introduction to Isolating RNA

  • Donald E. Macfarlane
  • Christopher E. Dahle
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 86)

Abstract

Ribonucleic acid (RNA) is an unbranched polymer of purine (adenine, guanine) and pyrimidine (cytosine, uracil) nucleotides joined by phosphodiester bonds.

1.1 Introduction

1.1.1 Structure

Ribonucleic acid (RNA) is an unbranched polymer of purine (adenine, guanine) and pyrimidine (cytosine, uracil) nucleotides joined by phosphodiester bonds.

The RNA polymer is bulkier than that of DNA (which lacks the 2′OH on the ribose). RNA is usually single-stranded, and it tends to form tertiary structures of high complexity, including hairpin loops, internal loops, bulges, and pseudo-knots (in which self complementary sequences align to form short, antiparallel double helical strands), and triple stranded structures (1, 2, 3). This complexity gives rise to functional molecules of much greater diversity than DNA, supporting the provocative concept that RNA evolved in the prebiotic era (4). Intact RNA is difficult to isolate because, as a long polymer, it is prone to mechanical or chemical degradation, and because of the existence of RNase, which is widely distributed on laboratory surfaces and difficult to destroy.

1.1.2 Function

It is increasingly recognized that RNA molecules act as enzymes as well as serving structural and informational functions. RNA within cells is generally associated with proteins and metal ions in ribonucleoprotein (RNP) complexes (5), of which ribosomes (which synthesize proteins), are the best-known example. RNA is synthesized in precursor form by transcription from DNA.

Transfer RNAs deliver amino acids to ribosomes, and consist of characteristic clover leaf structures about 80 nucleotides long. Many of the base residues are methylated or otherwise modified. Messenger RNA is capped at its 5′-end with methylated bases, and is usually polyadenylated at its 3′-end. Most eukaryotic mRNAs are spliced from then precursor transcripts to excise introns and juxtapose sequences from exons. mRNAs constitute about 1/l00 of the total RNA of the cell, and yet they carry all genetic information from DNA to the ribosome to generate appropriate sequences of amino acids in the synthesis of proteins. The half-life of mRNAs varies from a few minutes (in the case of eukaryotic regulatory protems) to years (in seeds and spores). Ribosomal RNA constitutes the bulk of cellular RNA, contributing three molecular species and about half the mass to the organelle which is assembled with more than fifty proteins. Heterogeneous nuclear RNA includes RNA undergoing processing by spliceosomes, in which RNA is itself catalytic.

1.2 Evolution of Methods for Isolating RNA

1.2.1 Purposes

The progress of scientific inquiry engenders a coordinated evolution of practical methodology and factual knowledge. As our understanding of RNA has progressed, the amounts and purity of RNA required for experiments has changed. Studies examining the infectivity of viral RNA, or the ability of an RNA to support translation in vitro, demand full-length RNA molecules, but urity (in the chemical sense) is less important than the elimination of interfering molecules. Analysis by ultracentrifugation or by gel electrophoresis and hybridization (Northern blots, typically requiring about 10 μg RNA) requires RNA with a high degree of preservation of polymer length, but these techniques are tolerate of gross impurity and occasional chemical modification of bases (seeChapters 11 and 13). Modern methods for measuring the quantity of an known RNA species present in a mixture (such as the RNase protection assay and branched chain analysis and needing up to 50 μg) are tolerant of both impurities and occasional strand breaks, but they require reproducible (and preferably quantitative) recovery of RNA. RNA isolated to prepare cDNA libraries require the highest degree of structural and sequence integrity. RNA intended for amplification by RT-PCR (typically less than 1 μg) must be free of inhibitors of reverse transcriptase or the DNA polymerase, but useful results can often be obtained with impure, degraded samples.

Special methods may be needed to prepare RNA from plants and single-cell organisms to rupture cell walls and to eliminate contaminating (non-nucleic acid) polymers. Methods to prepare RNA in a clinical area cannot employ noxious reagents, and should tolerate prolonged standing at room temperature. For some purposes it may be necessary to isolate RNA free of DNA. Almost all methods demand that the RNA product is free from RNase.

1.2.2 Early Methods

The earliest methods for isolating RNA were applied to viruses, such as the tobacco mosaic virus, in which the RNA is encapsulated in a protein coat. Brief heating of a suspension of purified viral particles resulted in a coagulum of denatured protein, and a solution of RNA, which was concentrated by dialysis and dried, yielding RNA with molecular weight ranging up to 200,000, a result which challenged the view that RNA was a prosthetic group for a (proteinaceous) enzyme (6). Work with eukaryote RNA was initially directed to subcellular fractions enriched for organelles consisting of ribonucleoproteins, such as ribosomes. During the subcellular fractionation, the RNA was retained in the ribonucleoprotein complex by maintaining a near-physiologic pH, ionic strength and divalent ion concentration, and the detergents used were either non-ionic or a low concentration of an anionic surfactant. These experimental conditions coincidently limited the disruption of the nucleus and the release of DNA. Subsequent differential centrifugation generally resulted in preparations of organelles containing RNA in high concentration. RNA in these purified organelles is relatively protected from RNase.

Following the preparation of the organelles, a variety of methods were used to dissociate the RNA from the protein, and these methods generally included an inhibitor of RNase. Anionic surfactants were particularly useful for this purpose, including sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (which, having a lower Krafft temperature, can be used in the cold (7), and sodium lauroylsarcosine (an amide derivative of SDS used because it is more compatible with cesium chloride centrifugation).

After the dissociation of RNA from the protein, the two can be separated by ultracentrifugation through a cesium chloride gradient (5.7M), a very useful technique which exploits the high buoyant density of RNA in cesium chloride (1.9), compared with that of DNA (1.7), and polysaccharides and glycogen (∼ 1.67) (8). Pure RNA is sedimented to the bottom of the tube.

Certain organic solvents can dissociate RNA from protein, and by exploiting the difference in hydrophobicity between RNA and protein, can separate them by generating two phases. The most commonly used reagent for this purpose is phenol. After phase separation, the RNA remains in the aqueous phase, whereas proteins (and DNA when conditions are appropriately adjusted) partition either into the phenol layer or collect at the interface.

Methods using phenol can be used to recover RNA directly from whole cells, without prior purification of nucleoprotein particles. In the methods described by Kirby (9), tissues were homogenized in phenol with m-cresol (added to reduce the melting temperature of the phenol and to improve its deproteinizing effect), 8-hydroxyquinoline (to chelate metal ions, reduce the rate of oxidation of phenol, and to assist in the inhibition of RNase), and nathphalene 1,5-disulfonate or sodium 4-aminosalicylate (as surfactants). The aqueous phase was collected, and repeatedly re-extracted with a phenol mixture, followed by precipitation of the RNA with ethanol plus either sodium acetate or sodium chloride/sodium benzoate/m-cresol. The yield of rapidly labeling RNA can be increased when the extraction with phenol is carried out at elevated temperature. The addition of chloroform to the phenol often increases the yield of RNA (10).

Methods employing phenol are widely used, but this obnoxious reagent causes much mischief in the hands of the unwary. Phenol (remarkably) does not completely inhibtt RNase, and it may actually disrupt the actions of other inhibitors of RNase. It is also prone to oxidize to reactive species which may degrade RNA, requiring that it be purified before use

1.2.3 Recent Methods

Chaotropic agents disrupt the forces responsible for cell structure. The use of guanidine hydrochloride (guanidinium chloride) led to the first preparation of eukaryotic RNA in highly polymerized form (11). When used at 4M, it inhibits RNase and dissociates nucleoproteins. The liberated RNA can be recovered by precipitation with ethanol, or by centrifugation (12).

Guanidine thiocyanate (guamdinium isothiocyanate) is a more powerful chaotrope, and is capable of dissolving most cell constituents, releasing RNA and inhibiting RNase. In the widely used method of Chirgwin (13), which can be applied directly to cells, tissues are homogenized in 4M guanidine thiocyanate, 0.1M -mercaptoethanol (a sulphydryl reductant), and 0.5% sodium lauroyl sarcosine. The resulting homogenate is layered on a cesium chloride gradient, and the RNA is ultracentrifuged overnight into a pellet. As an alternative to ultracentrifugation, the RNA can be precipitated with ethanol from the lysis solution, and repeatedly reprecipitated after redissolving in guanidine hydrochloride. The ultracentrifugation method is probably the most reliable method of obtaining high-quality RNA suitable for any purpose, but it is time consuming, and the number of samples that can be processed is limited by the availability of an ultracentrifuge.

The most commonly applied method for isolating RNA in the experimental laboratory uses a proprietary mixture of stabilized phenol and guanidine thiocyanate, into which the sample is homogenized. A chloroform reagent is then added to effect a separation of phases, during which RNA (but not DNA or protein) remains in the aqueous phase, from which it is precipitated (14).

We recently introduced a novel method for isolating RNA from whole cells which takes advantage of the properties of cationic surfactants. These interesting reagents have long been known to precipitate RNA and DNA from aqueous solution. We found that the completeness of this precipitation depended on the nature of the counter ion. We also found that appropriately selected cationic surfactants were capable of lysing cells efficiently, resulting in immediate precipitation of nucleic acids, presumably by the formation of reverse micelles (15). In this state, RNA is protected from RNase. The currently recommended procedure is to homogenize the cells in a solution of 0.1M tetradecyltrimethylammonium oxalate, followed by gentle centrifugation. After the supernatant is discarded, the pellet is extracting with 2M lithium chloride. RNA, being insoluble in this salt solution, remains in the pellet; but DNA, the surfactant, and some polysaccharides are solubilized. The RNA is then simply dissolved from the pellet with a buffer (16). This simple method avoids obnoxious reagents. Once the sample is mixed with the cationic surfactant, it can be mailed at room temperature to a reference laboratory. These two features are desirable for those planning to explore clinical applications of RNA-based diagnosis in a cost-sensitive age.

1.3 The Future

The improvements in techniques for isolating RNA that have occurred over the past two decades have materially advanced the progress of molecular biology. RNA isolation is becoming sufficiently reliable to envisage a huge growth in RNA-based diagnostic techniques. Once difficulties in this isolation of RNA have been overcome, RNA will be the most informative class of molecules in the clinical specimen, Informative RNA molecules are usually present in far greater number than the corresponding DNA. Like DNA, RNA reveals the genetic origin of the cell (or virus) containing it, and the analysis of RNA reveals additional information about the activity of the cell at the time that the specimen was collected.

Quite simple methods could be used to detect: invasion by pathogens, tumors with either gene rearrangements or expressing characteristic proteins, inherited disorders caused by altered expression of proteins, and diseases involving the synthesis of proteins characteristic of inflammatory responses. RNA-based methods are currently used to monitor the response to therapy of HIV and hepatitis C infections, and we can anticipate that a similar approach can be applied to a wide variety of disorders. In theory, even differential blood counts and blood typing can be performed using RNA-based methods.

As many readers will recall, isolating RNA used to be a frustrating and tedious task that was a prerequisite to experiments in molecular biology. The chapters in this volume eloquently attest to the advances in RNA isolation and manipulation that have been made over the years. Working with RNA is no longer the ogre it used to be!

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Copyright information

© Humana Press Inc. 1998

Authors and Affiliations

  • Donald E. Macfarlane
    • 1
  • Christopher E. Dahle
    • 2
  1. 1.Department of Internal MedicineUniversity of IowaIowa City
  2. 2.Department of MedicineUniversity of IowaIowa City

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