RNA Based

All protein is ultimately the product of mRNA coding information translated into amino acid sequences that constitute proteins, which then undergo a vast number of posttransla-tional modifications, resulting in more than a million identifiable proteins from fewer than 50,000 genes. In part, it is the potential to reduce this posttranslational complexity to manageable levels, as well as the precision and unambiguous result of RNA assays as compared to protein assays, which has driven the rapid development of RNA-based assays of interest to both biomedical research and clinical medicine. Nucleic acids are intrinsically more manageable; there are no issues with antibody specificity or ambiguous peaks on mass spectrometry. Even a single base is readily detected. Thus, many of the current methods for characterizing tumor or tissue gene expression use mRNA assays as surrogates for predicted protein gene product. Although it is true that mRNA assays fail to reflect the vast number of protein moieties that exist within a cell, many derived from the same gene, it is also true that all these proteins ultimately track back to mRNA message: if there is no message, there is no protein. The converse, of course, is not always correct: message may not result in protein, or the levels of protein may not parallel message levels, as a result of truncated or prolonged protein half-life, or even a failure of reliable translation without immediate proteasomal degradation, as occurs, for example, with mutated genes producing codon changes that result in premature stop codons and truncated protein. These are usually rapidly degraded, and in this case, abundant mRNA is not reflected in abundant detectable protein, which is rapidly degraded to undetectable peptides and amino acids. Despite these caveats, RNA assays are perhaps the most widely used assay for gene expression in cancer today. It is therefore useful to review the various methods used to identify and quantify mRNA expression.

RNA Blots (Northern Blot)

The oldest method to identify and roughly quantitate RNA is the Northern blot, named for its similarity to the DNA blot described by E.M. Southern.33 Many variations have subsequently been described, including simple transfer without electrophoretic separation of RNA samples to nitrocellulose or nylon filters followed by detection with a suitably specific radioactive or fluorescently labeled probe. A virtue of this method is its ability to both determine relative molecular mass and quantify the amount of mRNA present in the sample, based on density (or fluorescence) measurements of the hybridized, labeled probe. However, a major shortcoming is the large amount of RNA required, the need for extensive laboratory handling (purification, electrophoresis, etc.), and the time required for a result. Most human tissues, and especially tumor biopsies, rarely provide sufficient material for Northern blots. Consequently, alternative methods of RNA detection and quantitation have been developed.

RNA Amplification: Polymerase Chain Reaction a. Basic PCR: Polymerase chain reaction, or PCR, has become the method of choice for detecting even minute amounts of RNA or DNA, even in contaminated, impure, nb1 nb2 ES1 ES2 RMS RMS PNET

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