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Kits Take the Trickiness Out of RNA Isolation, Purification MINIMIZES CONTAMINATION: Ambion’s Rnase Zap destroys Rnase on laboratory surfaces. RNA is the most versatile biological molecule. It carries genetic information from the nucleus to the cytoplasm, then actively participates in protein synthesis; it also can function catalytically. All cells of a multicellular organism have similar collections of transfer and ribosomal RNAs, but it is the repertoire of messenger RNAs (mRNAs) that sculpts a specialized cell’s identity, expressing the subsets of genes that distinguish bone from muscle, xylem from phloem. So important is RNA that many investigators hypothesize that it played a pivotal role in the origin of life (see Research). Because RNA lies at the heart of gene expression, extracting it from cells is a common procedure in many cell biology, molecular biology, and biochemistry laboratories. "Obtaining high-quality, intact RNA is the first and often the most critical step in performing many fundamental molecular biology experiments, including northern blot analysis, nuclease protection assays, in vitro translation, reverse transcription PCR, and cDNA library construction,” says G. Russell Kimball, product manager at Ambion Inc. in Austin, Texas, a firm that calls itself "The RNA Company." RNA isolation has a reputation for being tricky. "When I train new graduate students or postdocs, the greatest challenge is overcoming their fear," notes Hugh Robertson, a professor of biochemistry at New York Hospital-Cornell Medical Center who studies RNA viruses. "They are taught in college courses that RNA is very difficult to work with, that it breaks down if you so much as look at it." The reason for the fear is the enzyme ribonuclease, or RNase for short, which chews up RNA. Any RNA isolation technique must remove RNase from experiments, workers, and surroundings. "The challenge in extracting RNA is that cells contain RNases in packets apart from RNA," says Robertson. "When you break a cell to extract the RNA, there is the possibility that the RNA will mix with RNase." RNases, with other intracellular digestive enzymes, are normally sequestered in lysosomes. These organelles are also known as the cell’s "suicide sacs" because when they are disrupted, their contents destroy the cell from within. Ambion Inc. Amersham Life Science Inc. AMRESCO Inc. Bio 101 Inc. Boehringer Mannheim Biochemical Corp. Clontech Laboratories Inc. CPG Inc. Epicentre Technologies Corp. 5 Prime -> 3 Prime Inc. Gentra Corp. Hybaid Ltd. Invitrogen Corp. Life Technologies Corp. Novagen Inc. PerSeptive Biosystems Inc. Pharmacia Biotech Inc. Promega Corp. Qiagen Inc. Sigma BioSciences Corp. Stratagene Corp. Since RNase can tolerate many methods of protein inactivation, such as autoclaving, most RNA isolation protocols use guanidine thiocyanate (GTC) to denature the troublesome enzyme. And because RNase from human skin can contaminate a specimen, gloves are a must when handling RNA. To minimize contamination, solutions are available to destroy RNase on laboratory surfaces. Ambion’s RNase ZAP is one such product. "Getting RNA out of cells presents a tough problem, because of RNases," agrees Olke Uhlenbeck, a professor of chemistry and biochemistry at the University of Colorado in Boulder, who wrote an article in the premier issue of the journal RNA (O. Uhlenbeck, RNA, 1:4-6, 1995) entitled "Keeping RNA Happy." One way to minimize RNase exposure is to prepare cells or tissues quickly. This may entail flash-freezing a sample in liquid nitrogen and grinding it up in a mortar and pestle, or using a device such as the Hybaid Ribolyser from Hybaid Ltd. in Middlesex, U.K. "The Ribolyser utilizes a simultaneous shaking and twisting motion at high speeds for cell disruption, which is caused by ceramic beads and detergents, which are provided in 2-milliliter tubes," explains Thomas Lernbecher, product manager for Hybaid. The instrument can process 12 samples at once. It breaks apart cells in seconds and is a component of RNA isolation kits offered by the company. MAGNETIC-BASED KIT: The mRNA Isolation Kit, available from Boehringer Mannheim Because RNA isolation and purification involve many reagents and steps, they present many opportunities to make mistakes or contaminate samples. Using commercially available kits may add cost but can save researchers time and increase yield and purity of extracted RNA. "One main reason to use a kit is the ease of use," says Ruth Schuman, president of Gentra Corp. in Minneapolis. "RNA isolation components are tricky to put together. Components have to be produced under good quality-control conditions, and it is time-consuming. A kit is very convenient." Adds Mary Ann Wiskerchen, technical services scientist at Promega Corp. in Madison, Wis.: "RNA kits for isolation of total RNA offer the assurance to customers that all the reagents have been qualified to be RNase-free. This is especially useful to scientists who do not perform many RNA isolations, as they do not need to put the effort into generating RNase-free solutions." VARIETIES AVAILABLE: Gentra’s PUREscript RNA Isolation Kits are distinguished by cell type. Many companies offer RNA isolation kits. The products differ in extraction of total RNA or mRNA; the type of tissues they work on; and whether they promise to optimize yield, speed, or purity. For example, 5 Prime -> 3 Prime Inc. of Boulder, Colo., offers six PERFECT RNA Total RNA Isolation kits. These come in three sizes (maxi, mini, and micro) for use on eukaryotic cells or on E. coli. Gentra’s PUREscript RNA Isolation Kits are distinguished by cell type. "We offer kits for whole blood and bone marrow; plant and animal [including human] tissue; tissue culture cells; and kits for bacteria and yeast," notes Schuman. "The kits are very similar. The blood kit has an extra component to eliminate red blood cells, and bacterial kits have specific cell suspension and lysis solutions." Most products work on cells or tissues directly, or isolate mRNA from already purified total RNA. Epicentre Technologies Corp. of Madison, Wis., offers an alternative, called the GELase Agarose Gel- Digesting Preparation. It extracts RNA that has already been separated by size on agarose gels. "A researcher can cut the gel, and use only the band of interest," explains Susan Bruni, product manager. "It is really easy to use. You just put the gel in a tube, add buffer and enzyme, and the RNA precipitates out." Isolating total RNA involves three steps: lysing cells, inhibiting RNase and getting rid of other proteins, and recovering intact RNA. A series of chemicals helps accomplish these tasks (P. Chomczynski, N. Sacchi, Analytical Biochemistry, 162:156-9, 1987). After cells are disrupted, GTC and sometimes beta- mercaptoethanol are added to inactivate RNase. N-lauroyl sarcosine is added to separate RNA from ribonucleoproteins. Then acidified phenol/chloroform/ isoamyl alcohol is added, which partitions RNA into an aqueous phase and DNA and protein into an organic phase. This step may be repeated to purify the fraction containing RNA. Finally, an alcohol such as isopropanol or ethanol is used to precipitate the RNA. Researchers have developed modifications to ease the procedure. Robertson, for example, adds DNase, which digests DNA, before the phenol extraction. This removes DNA early on. "Otherwise, when you break nuclei, the chromosomes pop out as a big, solid mess," he explains. "The DNA gets in the way of studying RNA." Products for total RNA extraction are generally variations on this organic extraction theme. TRIZOL reagent from Life Technologies Inc. of Gaithersburg, Md., for example, combines phenol and GTC, and the RNA Isolation Kit from Stratagene Corp. of La Jolla, Calif., combines the GTC and phenol/chloroform extraction steps. CPG Inc. of Lincoln Park, N.J., offers a Total RNA Isolation Kit that eliminates the alcohol precipitation step. USES GTC: The QuickPrep Total RNA Extraction Kit from Pharmacia Biotech includes lithium chloride. The ToTALLY RNA Total RNA Isolation Kit, from Ambion is also GTC/phenol/chloroform-based, but includes an optional step that uses the salt lithium chloride to remove DNA, carbohydrates, and heme groups. The QuickPrep Total RNA Extraction Kit from Pharmacia Biotech Inc. of Piscataway, N.J., uses GTC, but not phenol, and includes lithium chloride. Clontech Laboratories Inc. of Palo Alto, Calif., offers reagents in two solutions, called CLONsepA and CLONsepB, in its CLONsep Total RNA Isolation Kit. The first solution disrupts cells and precipitates RNA, and the second solution removes DNA and protein. Gentra’s PUREscript RNA Isolation Kits take a gentler approach than the GTC/phenol/chloroform method. "We use a nontoxic, proprietary, modified salt precipitation method, without guanidinium," says Schuman. "RNase inhibitors are in the reagents, and the salt method removes proteins. It is very gentle, so the RNA is of exceptional quality." Ambion’s new RNAqueous is a phenol-free RNA isolation product. Qiagen Inc. of Chatsworth, Calif., extracts total RNA using a unique spin column technology without organic extractions in its RNeasy Kits. Methods of extracting mRNA are based on the fact that these molecules have a string of adenines at one end, called a poly(A)+ tail. Two techniques use a string of 20 to 30 thymines, called oligo (dT), to bind the poly(A)+ tail and pull mRNAs out of mixtures. The difference between the two depends on whether solid supports or magnetic beads are used to extract the RNA. In one type of mRNA isolation protocol, the oligo (dT) is bound to a solid support. A series of washes and centrifugation steps removes other RNA species. Finally, bound RNA is eluted and collected. Most mRNA extraction kits based on a solid support use cellulose. "All kinds of materials can be used as a support. We opt for cellulose because the oligo (dT) has to move around in the solution in which the mRNA is dissolved," says Martin Kracklauer, technical services representative at Ambion. Some companies use steel beads, which are not as flexible, he maintains. Ambion’s Poly(A)Pure kits can be used on already isolated total RNA, or directly on eukaryotic cells or tissues. ISOLATES mRNA: Invitrogen’s FastTrack 2.0 kit contains pre-measured oligo (dT) cellulose powder. The FastTrack 2.0 mRNA Isolation Kit from Invitrogen Corp. of San Diego contains pre-measured oligo (dT) cellulose powder. It isolates mRNA from 0.4 g to 1 g of tissue, or 0.2 mg to 1 mg of total RNA. The company’s Micro-FastTrack kit targets samples of 10 mg to 200 mg of tissue or 100 µg of total RNA. Other mRNA isolation kits based on oligo (dT) bound to cellulose include Clontech’s mRNA Separator kit and 5 Prime ¤ 3 Prime’s Poly(A)+mRNA Isolation Kit. Also available along these lines are the QuickPrep Micro mRNA Purification Kit from Pharmacia Biotech Inc.; the Messenger RNA MicroIsolation Kit from Sigma BioSciences Corp. of St. Louis; the Poly(A) RNA Isolation Kit from Amersham Life Science Inc. of Arlington Heights, Ill.; and the Rapid mRNA Purification Kit from AMRESCO Inc. of Solon, Ohio. In the second approach to isolating mRNA, the oligo (dT) is linked to biotin. The biotinylated-oligo (dT) pulls out the poly(A)+ mRNA, and then paramagnetic particles labeled with streptavidin are added. A strong magnet attracts the paramagnetic particles, which attract the biotinylated-oligo (dT), which attracts the poly(A)+ RNA. After several washing steps, the mRNA is eluted by resuspending the magnetic particles in a buffer solution. FOR mRNA EXTRACTION: Promega’s PolyATtract mRNA Isolation System 1000 extracts mRNA from total RNA. It works on tissues directly. Promega’s PolyATtract mRNA Isolation Systems extract mRNA from total RNA using MagneSphere Streptavidin Paramagnetic Particles, in a device called a Magnetic Separation stand. The PolyATtract System 1000 works on tissues directly, and the PolyATtract Series 9600 works in a 96-well format. "You can watch it happen. A brown solution clears, and brown particles become plastered against the side wall. You pour the supernatant off, add buffer, and it goes back into solution. If you do this three times, there is really almost a zero probability that there is anything but specifically bound mRNA," claims Mark Lepinske, technical services scientist at Promega. The Hybaid Recovery mRNA Kit also uses magnetic beads and works on total RNA or directly on animal cells or tissue culture cells. CPG’s MPG Direct mRNA Purification Kits use the magnetic porous glass system to extract mRNA from animal and plant tissues, and the MPG mRNA Purification Kit works on purified total RNA. The glass particles can be reused for multiple rounds on the same sample, which maximizes yield. GOOD ON PLANT CELLS: Novagen’s Straight A’s mRNA Isolation System The mRNaid Kit from Bio 101 Inc. of La Jolla, Calif., skips biotin/streptavidin binding by attaching magnetic beads directly to biotin. One adds this to RNA, incubates on ice for 10 to 30 minutes, applies the magnet, washes the beads, and elutes the RNA. The Straight A’s mRNA Isolation System from Novagen Inc. of Madison, Wis., works particularly well on hard-to-crack plant cells and tissues, such as cotton fibers and soybean leaves. Other magnetic-based kits include the BioMag mRNA Purification Kit from PerSeptive Biosystems Inc. of Framingham, Mass., and the mRNA Isolation Kit from Boehringer Mannheim Biochemical Corp. of Indianapolis. Isolating RNA, like collecting any molecule from biological material, is difficult and the yield is never completely pure because of the chemical complexity of cells. A strategy to vastly improve purity is to synthesize RNA of interest in vitro—a little like cloning a peanut, rather than extracting it from a candy bar. "We can make RNA in vitro with RNA polymerase to synthesize RNA from DNA, and then remove the enzyme," states Uhlenbeck. "There are many strategies to remove the RNA polymerase, but using phage T7 RNA polymerase is the most common. It can be separated out with an ion-exchange column. This is starting to be a way for people to avoid using denaturing agents to obtain RNA." Biochemists pioneered in vitro transcription, which is being used increasingly by molecular biologists, he adds. Laura Landweber, an assistant professor in the department of ecology and evolutionary biology at Princeton University, uses in vitro transcription to produce enormous numbers of different RNA molecules from DNA pieces made with a DNA synthesizer. She studies novel RNA catalytic functions that might have been important when nucleic acids and proteins first interacted to form life. "We generally synthesize RNA by in vitro transcription using T7 RNA polymerase from PCR templates, and we purify the RNA by precipitation or gel purification," she explains. "The RNA pools are constructed as large collections of synthetic DNA molecules and then transcribed into RNA." Adds Robertson: "In vitro transcription provides synthetic RNA you’d otherwise have to isolate. It can be scaled up and overcomes the difficulties of nucleases." But for the many researchers who use the organic extraction approach to obtain RNA, there are a great variety of kits from which to choose. Ricki Lewis, a freelance science writer based in Scotia, N.Y., is the author of several biology textbooks. She is online at 76715.3517@compuserve.com.

Kits Take The Trickiness Out Of RNA Isolation, Purification

Mar 30, 1997

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