The utility of DNA- or RNA-based testing depends, in large part, on the quality and nature of the diagnostic sample. A variety of methods is available to extract nucleic acids for analysis. The choice of technique should consider both the sample source and the nature of the eventual assay. Both DNA and RNA can provide valuable clinical information. Genotype analysis and infectious disease testing represent two of the primary clinical uses of DNA, but it can also be used for less obvious applications like the assessment of bone marrow transplant engraftment.
The ability to purify and test RNA provides additional and important clinical data. Purified mRNA, for example, can reveal gene expression patterns. In the last few years, two distinct types of B-cell lymphoma have been separated on the basis of their respective gene expression patterns (1). Indeed, molecular classification of malignancy based on gene expression profiles is a promising and rapidly growing field. On a simpler level, the expression pattern of a single gene might be important. The BCR/abl fusion product seen in most cases of chronic myelogenous leukemia (CML) can be identified by reverse transcriptase-polymerase chain reaction (RT-PCR) performed on a purified RNA sample. RNA testing is also important in the detection and quantification (viral load) of retroviral infection. The utility of RNA is, unfortunately, counterbalanced by the inherently labile nature of RNA and seemingly ubiquitous presence of RNase. Nucleic acid degradation is always a concern, and RNA is especially vulnerable.
Each tissue source and extraction method presents its own potential quality assurance issues. Similarly, the needs of the eventual assay could vary. Whereas some techniques require high-molecular-weight nucleic acid (Southern blots, pulsed-field gel electrophoresis), others (including PCR-based protocols) often work well with smaller fragments. In addition to nucleic acid quality, purity and concentration are important factors to consider. Many testing techniques are sensitive to contaminating protein, lipopolysaccharide, or tissue preservative. More obvious is the need for adequate concentrations of nucleic acid. There are numerous methods for detecting and quantifying nucleic acid, with varying degrees of sensitivity and background noise. Testing based on DNA fluorescence, for example, might require higher nucleic acid concentrations than that based on radioimaging. These three parameters—quality, purity, and concentration—can be optimized with careful selection of sample source and technique.
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