OVERVIEW

Fluorescent cDNA microarray technology is useful for making estimates of the abundance of particular messages relative to a designated source of mRNA that serves as a reference point. Commercial support of this technology has recently reached a level where it is reasonable for departments or large laboratories to consider setting up their own cDNA array facility. This set of protocols is intended to serve as a basic introduction to making and using cDNA microarrays for those embarking on this path. There are three fundamental types of operations required in a cDNA microarray experiment. The first operation, BASIC PROTOCOL 1, cDNA AMPLIFICATION AND PRINTING, deals with making the cDNA microarray itself. It is necessary to collect an inventory of cDNA bacterial clones that represent the genes whose message abundance you wish to survey. Plasmid templates are made from these clones and used as PCR substrates to produce DNA representations of the EST inserts. The PCR products are then purified and spotted onto poly-L-lysine coated microscope slides. In the second operation, BASIC PROTOCOL 2, RNA EXTRACTION AND LABELING, RNA is extracted from the cell samples to be examined, purified, and used as the substrate for reverse transcription in the presence of fluor-derivatized nucleotides. This procedure provides the tagged representations of the mRNA pools of the samples that will be hybridized to the gene-specific cDNA detectors immobilized on the microarray. The third fundamental operation, BASIC PROTOCOL 3, HYBRIDIZATION AND DATA EXTRACTION, covers the steps in which fluor-labeled cDNAs hybridize to their complements on the microarray, and the resulting localized concentrations of fluorescent molecules are detected and quantitated.