^ transcription mRNA
Southern blot sequencing WAVE SNP chips
Northern blot RT PCR QRTPCR DNA Microarrays
Immunohistochemistry ELISA Western
figure 21.1. Diagram: DNA to RNA to protein.
amplification is most significantly associated with prognosis in breast cancer, because there is a close association between amplification and elevated protein expression, it is generally sufficient to simply detect the protein by IHC. However, there is significant disagreement as to the superiority of IHC (e.g., protein detection) as opposed to FISH (fluorescent in situ hybridization to detect DNA copy number), and whether DNA amplification is more important than protein expression. A similar controversy surrounds childhood neuroblas-toma, where MYCN amplification detected by FISH is the standard diagnostic procedure for MYCN determination; polymerase chain reaction (PCR) for elevated MYCN expression levels is occasionally misleading and not a reliable marker for genomic amplification. Similarly, detection of MYCN protein by IHC is not a reliable surrogate either.
Despite these potential limitations, IHC is far and away the most common technique employed to augment morphologic diagnosis. It is enjoying something of a resurgence, as genes detected by other methods (as described next) need to be validated at the protein level. To serve this burgeoning need, innumerable biotechnology companies producing vast numbers of antibodies specific for a given gene product have arisen and increasingly supply the specific antibody paired with the gene of interest. Two benefits accrue: protein staining is considered the diagnostic gold standard (as not all expressed genes necessarily result in expressed protein), and the technology is simple, cheap, reproducible (with some caveats), and technically straightforward.
Western Blots and Immunoprecipitation
Historical problems with nonspecificity of antibody staining on tissue sections, coupled with a relative lack of quantita-
tion, led to the development of a method to detect protein and confirm identity based on objective criteria such as molecular mass and reactivity with an allegedly specific antibody. In this method, the crude protein extract is incubated with an antibody and the antigen-antibody complex is precipitated from solution, then separated into antigen and antibody by gel electrophoresis. Conversely, the mixture can be electrophoresed and separated by molecular mass, then transferred to a filter membrane (the "Western" blot, as opposed to a DNA Southern blot or RNA Northern blot) and incubated with the antibody. Either way, the protein can be identified by its reactivity with antibody and its relative mass, or molecular weight. An example of a comparative Western analysis of the same protein from a series of Ewing's tumors is illustrated in Figure 21.3. It is immediately apparent that the relative amount of the protein in erstwhile identical tumors is in reality markedly different, thereby documenting a quantitative difference in protein content that would be difficult to assess by IHC, for example, where only
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Among the evils which a vitiated appetite has fastened upon mankind, those that arise from the use of Tobacco hold a prominent place, and call loudly for reform. We pity the poor Chinese, who stupifies body and mind with opium, and the wretched Hindoo, who is under a similar slavery to his favorite plant, the Betel but we present the humiliating spectacle of an enlightened and christian nation, wasting annually more than twenty-five millions of dollars, and destroying the health and the lives of thousands, by a practice not at all less degrading than that of the Chinese or Hindoo.