Appendix M Advanced Analysis Techniques 1 DNA DNA analysis requires relatively little tissue (polymerase chain reaction [PCR] amplification techniques require only a few nanograms). A milligram of tissue left over from diagnostic procedures would be sufficient to enable almost infinite amplification and distribution. Since DNA sequences are 99.9-percent identical, it is possible to consider assaying for just differences, such as single nucleotide polymorphisms (SNPs). There are a variety of technologies that enable high-throughput serial analysis of SNPs, or even parallel analysis of a limited number of SNPs. The challenge with scaling these technologies to whole-genome SNP analysis is their requirement for specific primers for each SNP, a costly and complex proposal. It is possible to employ a generic approach for reducing the complexity of the whole genome and to perform a parallel analysis of 10,000 SNPs on a DNA microarray. This level of genetic information could be useful for analyses like loss-of-heterozygosity studies in cancer. As the DNA resource is close to inexhaustible, tissue still would be available when more powerful whole-genome SNP analysis is developed, likely in the near future. Protein Other than using two-dimensional gels, there is no way to do whole-proteome analysis. Welljustified specimen use for specific proteomic studies makes the most sense today. In the future, technologies may allow researchers to look at transcriptomes (noncoding regions that are transcribed), which may provide new targets for therapeutic intervention. Therefore, it would be wise to preserve specimens for proteomic analysis when more procedures become available. RNA Reverse transcription-PCR and Northern blot analyses are useful for identification of a limited number of RNA markers but do not scale effectively for whole-genome analysis and should be reserved for hypothesis testing. Microarrays for whole-genome expression analysis are achievable and align with the goal of acquiring the maximum amount of information possible from the specimens. Whole-genome expression analysis using microarrays is now a standard approach in both the pharmaceutical and academic sectors. Microarrays can be used to determine if a tumor is benign or malignant, guide therapeutic choices, identify new classes of tumors, and predict patient outcome; they also have many other potential uses. With currently available technologies, 0.5 to 1 mg of tissue is needed to generate total RNA for whole-genome microarray analysis. It also is possible to recover labeled cRNA with certain technologies and save this tissue for future analysis.

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Footnotes 1 Source: Technology Platforms presentation by Dr. Thane Kreiner, Affymetrix, Inc., NDC Forum II, March 6, 2003.