The Human Genome Project promises to provide us with a molecular fingerprint of an individual. Pursuit of this ultimate goal has been the impetus for additional benefit to science: advancement of technology that provides powerful tools used in the analysis of gene expression.
High-throughput or array technology is continually being refined, providing us with important descriptive information of disease processes through mRNA-based analyses of gene expression. However, it is becoming clear that gene-based analysis is not sufficient for complete understanding of the phenotypic expression at the molecular level. The field of proteomics, in which the protein structure and function are determined from the genetic blueprint, is the next frontier that may add substantially to our understanding and management of diseases. With the advancement in high-throughput technology, rigorous demands are being placed on the procurement of tissue and providing of homogeneous cell populations, a circumstance that has lead to the development of tools such as microarray technology and laser capture microdissection (LCM).
Microarray Technology
Microarray technology, also known as “genechip” technology, has been an area of great interest particularly in differential analysis of gene expression. It allows for rapid quantitative measurement of gene expression in a tissue sample that is of interest on a large scale. Its advantage over blot techniques is its high degree of automation, parallelism and resolution. This technology takes advantage of the highly selective binding of nucleic acid molecules to complementary sequences.
The chip is composed of large numbers of DNA probes repeated in each location of the array. The probes may include the genome of an entire organism (or a subset of interest) which then is deposited on a glass or other material surface. The use of robotics has automated this process and increased its efficiency and consistency. RNA derived from the tissue is then added to the surface of these chips and then binds to the complementary DNA probes on the chip. The result is a highly parallel sorting process, and the binding can be quantified using a laser to detect the fluorescence-labeled RNA sample. Although the principle is not new, the high degree of automation and parallelism afforded by this technique has revolutionized biomedical discovery. A detailed review of DNA microarray gene expression analysis technology, including its applications to neurological disorders and its limitations, is available in Steven A. Greenberg’s article published in Neurology (2001).
Laser Capture Microdissection
LCM, a relatively new technology developed by the National Institutes of Health, has been commercially available through a collaborative effort with Arcturus Engineering Inc. LCM is a method for procuring cells from specific microscopic regions of tissue sections. Its advantage is the reproducibility and accuracy of selecting specific cells from complex tissue samples for subsequent analysis. Cells of interest are identified by microscopy and targeted. A variety of immunohistochemical stains can be used to identify cell populations of interest and thin sections of tissue can be fixed and embedded in paraffin or frozen. At the push of a button, a laser beam focally activates a special transfer film which bonds specifically to the cells targeted. The transfer film with the bonded cells is then lifted and separated from the unwanted cells. This process provides a homogeneous sample of cells that can then be used to procure DNA, RNA or protein.
There has been an explosion in the use of these powerful technological tools in basic science research, and for neurological disorders they are being used to study brain tumors, neuropathies and myopathies. Differential gene expression analysis between diseased and normal tissue is providing insight into the molecular pathogenesis of these neurological disorders. In addition to molecular phenotypinng (expression profiling), other applications for neurological disorders include functional genomics (gene function in gene regulatory networks), pharmacogenomics (drug development and prediction of efficacy and toxicity) and developmental biology (gene function in the control of development).
As the performance and technical issues of LCM and expression arrays are being refined, large amounts of data are being generated, giving birth to the field of bioinformatics-finding solutions to biological problems using computer science methodology such as knowledge representation, data storage and retrieval and database management. As the sequencing of the human genome nears completion, advances in proteomics and bioinformatics may ultimately provide a wealth of information to elucidate the pathophysiology of neurological diseases and facilitate the development of effective therapies.
Prithvi Narayan, MD, is a resident at Emory University School of Medicine, Atlanta, Ga.