Indexed on: 01 Jan '01Published on: 01 Jan '01Published in: Methods in molecular medicine
Identification of alterations in gene expression is an important step in understanding the development and progression of human disease. For pigmentary disorders with an unresolved hereditary component, genetic and epigenetic changes that alter the expression of genes as a direct or indirect consequence can be investigated by expression analysis. This can be of particular importance in conditions in which multiple genes are involved, as in melanoma or vitiligo (1,2). Differential display analysis is a technique that is widely used and entails semiquantitative polymerase chain reaction (PCR) amplification of 3' ends of messenger RNA (mRNA) (3- 6). The primer sets used hybridize to the poly-A tail of mRNA on one end, and to an arbitrary countersequence on the other. Each set of primers is designed to amplify some 150 messages present in the RNA under study. These messages are subsequently separated by polyacrylamide gel electrophoresis. Using a labeled nucleotide in the PCR reaction, the intensity of each band can subsequently be compared in samples of interest. Following reamplification, confirmation, cloning, and sequencing, the fragments of interest are further analyzed. Figure 1 outlines this sequence of events. Although the length of the fragments amplified usually excludes coding regions, the 3' noncoding regions are unique and can serve to identify the gene of interest, provided the fragment belongs to a gene or an expressed sequence tag (EST) that has previously been submitted to GenBank (7).