DNA is the repository of genetic information, which is then transcribed into RNA. RNA forms ribosomes, transfer RNA (tRNA), and messenger RNA (mRNA) for protein translation, and is used for other special functions in the regulation of gene expression that have only recently been discovered. DNA is a hardy molecule present at stable cellular levels, with such well-known exceptions as the amplification and deletion of genes in tumor cells. By contrast, the level of RNA corresponding to a gene can fluctuate dramatically within a very short time in response to changes in the cell's microenvironment and functional needs. This fluctuation results from changes in both the rate of transcription and degradation of an RNA.
DNA is relatively easy to isolate and store because deoxyribonucleases (DNases) are easily denatured by heating or inhibited by sequestration of divalent cations. RNA, by contrast, is rapidly degraded by a variety of ribonuclease (RNase) enzymes that are replete within the cell and on the skin surface. While this is necessary for cellular homeostasis, the ubiquity of RNases leads to a problem for the molecular analysis of RNA. RNases are very stable, active in virtually any aqueous environment, and can regain their activity after denaturation. As a result, RNA is subject to rapid degradation by RNases in most laboratory settings, making RNA a notoriously labile molecule. The rate of degradation varies among RNA species, leading to further analytical complexity.
RNA analysis depends on successful RNA isolation and preservation. The overall techniques are similar to those described above for nucleic acid isolation, but with the mandatory addition of steps to inhibit or degrade cellular RNases and prevent their reintroduction into the isolated RNA. RNA isolation must be performed promptly after specimen collection, particularly if quantitation of RNA is desired. If RNA isolation is delayed, the sample should be stored at -80°C, or at an intermediary isolation point in a stable buffer with RNase inhibitors. Scrupulously clean laboratory technique is required, including careful cleansing of laboratory equipment with bleach, autoclaving of glassware, preparation of reagents with nuclease-free water, and wearing gloves with frequent glove changes. Addition of GITC or beta-mercaptoethanol to the RNA isolation reagents will inhibit or denature RNases present in the sample. The isolated RNA is rehydrated in water or TE buffer that is nuclease free, and stored at -80°C to further inhibit the activity of any residual RNases.
Despite these difficulties, RNA is valuable in the clinical molecular laboratory for several reasons. Many clinically significant viruses such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV) have RNA genomes. Quantitation of RNA provides an important measure of gene expression, which can be used in the diagnosis or monitoring of disease. In addition, mRNA does not contain introns, which is an advantage when analyzing neoplastic translocations with variable intronic breakpoints such as BCR-ABL.5
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