DNA microarrays are created by robotic machines that arrange minuscule amounts of hundreds or thousands of gene sequences on a single microscope slide. Researchers have a database of over 40,000 gene sequences that they can use for this purpose. When a gene is activated, cellular machinery begins to copy certain segments of that gene. The resulting product is known as messenger RNA (mRNA), which is the body’s template for creating proteins. The mRNA produced by the cell is complementary, and therefore will bind to the original portion of the DNA strand from which it was copied.
To determine which genes are turned on and which are turned off in a given cell, a researcher must first collect the messenger RNA molecules present in that cell. The researcher then labels each mRNA molecule by using a reverse transcriptase enzyme (RT) that generates a complementary cDNA to the mRNA. During that process fluorescent nucleotides are attached to the cDNA. The tumor and the normal samples are labeled with different fluorescent dyes. Next, the researcher places the labeled cDNAs onto a DNA microarray slide. The labeled cDNAs that represent mRNAs in the cell will then hybridize – or bind – to their synthetic complementary DNAs attached on the microarray slide, leaving its fluorescent tag. A researcher must then use a special scanner to measure the fluorescent intensity for each spot/areas on the microarray slide.
If a particular gene is very active, it produces many molecules of messenger RNA, thus, more labeled cDNAs, which hybridize to the DNA on the microarray slide and generate a very bright fluorescent area. Genes that are somewhat less active produce fewer mRNAs, thus, less labeled cDNAs, which results in dimmer fluorescent spots. If there is no fluorescence, none of the messenger molecules have hybridized to the DNA, indicating that the gene is inactive. Researchers frequently use this technique to examine the activity of various genes at different times. When co-hybridizing Tumor samples (Red Dye) and Normal sample (Green dye) together, they will compete for the synthetic complementary DNAs on the microarray slide. As a result, if the spot is red, this means that that specific gene is more expressed in tumor than in normal (up-regulated in cancer). If a spot is Green, that means that that gene is more expressed in the Normal tissue (Down regulated in cancer). If a spot is yellow that means that that specific gene is equally expressed in normal and tumor.