Methods: cDNA Arrays

How arrays work.

There are about 100,000 human genes and the complete set occurs in nearly all cells of our body. When genes are "working" a partial copy of the genetic material, i.e. the DNA of the gene, is produced and this partial copy is called messenger RNA (mRNA). The mRNA can be detected and so tells us when a gene is working.

Figure 1: The arrayer has a number of pins that are first dipped into the well plate, drawing out a small bit of each solution on each pin (a). The pins then "print" the chip by releasing the solution onto a glass slide (b). The gene chip (c) now contains spots, each containing the DNA for one gene.
Credit: Barbara Aulicino/ American Scientist. Hamadeh and Afshari, American Scientist, 2000; 88:510. Used with permission.

In prostate cancer, some genes are working when they should not be and conversely some genes fail to work when they should. These changes show up as altered amounts of mRNA compared to normal cells and constitute the "profile" that we seek. The mRNA will bind to DNA of its own gene and that is the key to detecting when genes are working. Representative portions of DNA from a single gene can be immobilized as tiny spots on glass surfaces using "robots" (Fig. 1).

Figure 2: Selected DNA Microarrays (a) Oligonucleotide array synthesized in situ with photochemical technology by Affymetrix. (b) Oligonucleotide array synthesized in situ with ink-jet technology (Image courtesy of Rosetta Inpharmatics). (c) DNA microarray printed on a glass slide.
Reprinted from Cell, Young, "Biomedical Discovery with DNA Arrays", 9-15, Copyright 2000, with permission from Elsevier Science

The position of immobilized DNA is different but known for each gene. The small spot size means hundreds and of gene representatives can be placed on a surface thus making an array. These spots will bind and hold mRNA that has been extracted from tumor only if the corresponding gene in the tumor was working or causing mRNA to be made. The mRNA from a tumor is first processed to make it fluoresce and then mixed with a fluorescent "reference" preparation for comparison purposes. The mix is then applied to the glass surface with all gene representations available.

The red colored spots in Fig. 2 indicate that the gene represented at the "fluorescing positions" was more abundant than in the reference sample while yellow spots indicate the reverse. If the reference material is from normal tissue, the comparison provides direct information about which genes are working or not in prostate cancer.

Figure 3: Preliminary Results

Preliminary Results. A small example of results from studies at SKCC is shown in Fig. 3. Here the expression of 159 genes are represented as "bars" for two cell lines commonly studies in the laboratory and one human prostate tumor. The genes that are represented here are not labeled but the "tree" at the top indicates that the bars (genes) are ordered so that similar "behaving" genes in each of the three samples are next to each other. The while line under one patch of bars shows an area where the amount of mRNA for several genes is very different tumor compared to cell lines and may indicate that particular "gene expression" distinctions of prostate tumors are emerging.

Figure 4: Professors Dan Mercola, MD, PhD and Michael McClelland, PhD, with array robot.

Array making. At SKCC we are currently preparing arrays for this study arrays with over 5,000 human genes represented and expect to make arrays of nearly 40,000 within a year. The current arrays are utilized by the UCSD Cancer Center for the analysis of prostate cancer specimens collected by the SKCC and USCD Cancer Center cooperating tumor banks.

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