Cell LysisLysis (Genetics & Inherited Conditions)
RNA isolation is a difficult proposition. RNA has a short life span in cells (as short as minutes in bacteria), and it is somewhat chemically unstable. In addition, enzymes that degrade RNA (RNases) are widespread in the environment, further complicating the task of separating intact RNA from other molecules in the cell.
The first step in RNA isolation is rapidly breaking open cells under conditions where RNA will not be degraded. One method involves freezing cells immediately in liquid nitrogen, then grinding the cells in liquid nitrogen in order to prevent any RNA degradation. Other methods involve lysing cells in the presence of strong protein denaturants so that any RNases present in the cell or the environment will be rapidly inactivated. The difficulty of the cell lysis step depends substantially on the type of cell involved. Bacterial and fungal cells are typically much more difficult to break open than cells from mammals. As a consequence, it is often more difficult to isolate intact RNA from bacteria and fungi.
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Protein Denaturation and Further Purification (Genetics & Inherited Conditions)
The next step in RNA isolation is to denature all proteins from the cell, to ensure that RNases will be inactive. In many cases, this is done at the same time as cell lysis. RNases are among the most resilient enzymes known, capable of being boiled or even autoclaved, yet retaining the ability to cleave RNA once they cool down. Consequently, the RNA next needs to be separated from RNases and other proteins to ensure that it will remain intact.
The separation of RNA from the rest of the macromolecules in the cell can be accomplished in a number of ways. One of the older methods for purifying RNA uses ultracentrifugation in very dense cesium chloride solutions. During high-speed centrifugation, these solutions create a gradient, with the greatest density at the bottom of the tube. RNA is the densest macromolecule in the cell, so it forms a pellet in the bottom of the ultracentrifuge tube. A newer technique for RNA purification involves the use of columns that bind RNA but not other macromolecules. The columns are washed to remove impurities, such as DNA and proteins, and then the RNA is eluted from the column matrix. A newer technique is based on the observation that, at an appropriate pH (level of acidity), RNA partitions into the water phase of a water-organic mixture. DNA and proteins either are retained at the boundary of the water-organic mixture or are dissolved in the organic phase....
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Special RNA Isolation Procedures (Genetics & Inherited Conditions)
In some cases, a geneticist wants to isolate only RNA from the cytoplasm of the cell, since RNA from the nucleus may be more heterogeneous. In this case, cells are lysed using a gentle detergent that disrupts the cytoplasmic membrane, without disturbing the nuclear membrane. Centrifugation is used to separate the nuclei from the cytoplasm and then the cytoplasmic RNA is further purified as described above.
For some procedures, such as RT-PCR, the RNA sometimes needs to be further purified to ensure that no contaminating DNA is present. In this case, the RNA sample may be treated with the enzyme DNase I, which destroys DNA but leaves RNA intact.
For other procedures, like cDNA library construction, the RNA is often purified to remove ribosomal RNA (rRNA), transfer RNA (tRNA), and other stable RNAs, since the majority of RNA in the cell (typically more than 90 percent) is rRNA and tRNA. In this case, the RNA solution is treated by incubating it with single-stranded DNA containing a chain of eighteen to twenty thymine nucleotides, either on a column or in solution. Messenger RNA (mRNA) from eukaryotes contains runs of twenty to two hundred adenine nucleotides that bind to the single-stranded DNA and allow the mRNA to be purified away from the stable RNAs.
Like most techniques in genetics, RNA isolation methods have improved greatly over the years. With advances in methods for studying gene...
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Further Reading (Genetics & Inherited Conditions)
Ausubel, Fredrick, et al. Current Protocols in Molecular Biology. Hoboken, N.J.: John Wiley and Sons, 1998. Includes RNA isolation protocols from several different laboratories.
Avison, Matthew B. “Isolation and Analysis of RNA.” In Measuring Gene Expression. New York: Taylor & Francis, 2007. Discusses the properties of different types of RNA and various methods of isolating, purifying, and stabilizing RNA. Describes six protocols for isolating RNA from animal cells and tissues, bacterial and yeast cells, plant and filamentous fungal cells, and tissue culture cells.
Clark, David P. “Nucleic Acids: Isolation, Purification, Detection, and Hybridization.” In Molecular Biology. Boston: Elsevier Academic Press, 2005. Includes information about RNA isolation.
Farrell, Robert E., Jr. RNA Methodologies: A Laboratory Guide for Isolation and Characterization. 3d ed. Boston: Elsevier/Academic Press, 2005. Probably the definitive book on RNA techniques, including RNA isolation. Includes a substantial amount of background information, as well as detailed protocols.
Liu, Dongyou, ed. Handbook of Nucleic Acid Purification. Boca Raton, Fla.: CRC Press, 2009. Describes various nucleic acid isolation methods, with sections focusing on techniques to analyze viruses, bacteria, fungi, parasites, insects, mammals, and plants.
O’Connell, Joe, ed. RT-PCR...
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Web Site of Interest (Genetics & Inherited Conditions)
Ambion/Applied Biosystems, The Basics: RNA Isolation. http://www.ambion.com/techlib/basics/rnaisol/index.html. Ambion, a division of Applied Biosystems, manufactures products for scientists conducting RNA research. Its Web site provides an illustrated description of the RNA isolation process with links to an accompanying article, “Cell Disruption: Getting the RNA Out.”
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